Antibodies targeting b-cell receptor complex membrane bound igm and uses thereof

ABSTRACT

The present invention relates to antibodies targeting the membrane bound IgM (mIgM) of the B-cell receptor complex found in B-cell lymphomas and leukemias and uses thereof. Another aspect of the present invention is the use of anti-B-Cell mIgM antibodies in the treatment of Be-cell malignancies, including B-cell lymphomas and leukemias.

GOVERNMENT RIGHTS

This invention was made in part using government support under SBIRGrant No. 1 R43 A1081332-01A1 awarded by the National Institutes ofHealth. The government has certain rights to this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 1, 2014, isnamed 10199-003571-WO0_SL.txt and is 7,207 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies targeting the membrane boundIgM (mIgM) of the B-cell Receptor Complex found in B-cell lymphomas andleukemias and uses thereof.

BACKGROUND OF THE INVENTION

B-cell malignancies comprise the major subtype of lymphomas today withover 100,000 new cases per year. The vast majority of patients are notcurable despite the apparent sensitivity of these diseases to a numberof drugs and biologic agents commonly in use. A characteristic of manyB-cell lymphomas and leukemias is that chemotherapy and/or biologicbased responses are readily obtainable, but cures are more difficult.B-cell lymphoma can be a very aggressive disease where many of thepatients do not respond to conventional treatment. It is apparent thatresidual clones of neoplastic cells remain after log cell kill withchemotherapy and/or biologic therapies. The ability to cure thesediseases will be dependent on the success of eradication of all tumorcells, especially tumor stem cells. Anti-CD20 antibodies such asrituximab, ofatumumab, obinutuzumab, and tositumomab, have also beenused to treat B-cell derived malignancies as single agents, aspotentiators of chemotherapy, as maintenance therapy and as vehicles todeliver radioisotopes/drugs. These antibodies bind to CD20, a restrictedB-cell differentiation antigen expressed only by normal and malignantB-cells. Because of the expression of CD20 antigen on both normal andmalignant B-cells, anti-CD20 antibodies can also lead to the destructionof a portion of normal B-cells, the long term consequences of which areunknown. (See, Smith M R, Oncogene 22:7359-7368 (2003); Jacobs S A, etal., Expert Opin Bio Ther 7:1749-1762, (2007)).

In contrast to CD20, the B-cell Receptor Complex (BCRC) is the centraldifferentiation signaling element of the B-cell arm of the immune systemand this molecule is expressed on the surface of all B-cellmalignancies. The BCRC comprises a cell-surface membrane bound Ig (mIg)(such as mIgM, mIgG, mIgA, mIgE and mIgD) and a closely associatedco-signaling molecule CD79αβ. Previous strategies to target the BCRCmolecules in B-cell malignancies have focused on the unique CDRsequences specific for each monoclonal tumor. (See, Miller R A, et al.,N Engl J Med 306:517, (1982); Levy R, et al., J Natl Cancer InstMonographs 10:61 (1990); Davis T A, et al., Blood 92:1184-1190 (1998)).However, as a consequence of the uniqueness of each CDR, this approachnecessitated the generation of a specific drug for each patient, whichproved not to be feasible in the clinic. These early clinical studiestargeting the BCRC did, however, demonstrate anti-tumor activity.

The B-cell Receptor (BCR) initiates a driver pathway in B-celllymphoma-leukemia. One strategy has been to target BCRC associatedcytoplasmic molecules such as the Syk tyrosine kinase, a downstreammediator of the BCRC signaling pathway, to inhibit downstream pathwaytyrosine kinases. Both vertical and horizontal membrane BCRCinteractions render this downstream pathway complex and redundant. Asthe Syk tyrosine kinase pathway is not restricted to B-cell lineagetissue, its inhibition leads to unwanted immune effects, possiblepro-oncogenic effects in breast tissue and other toxicities innon-hematopoietic cells. Further downstream of the BCRC is the Brutontyrosine kinase (BTK). Bruton tyrosine kinase inhibition has alsoemerged as a compelling target downstream of the BCR, which is now anapproved strategy through the utilization of the drug ibrutinib. Theapproval of ibrutinib, the first BTK inhibitor demonstrating potentactivity, provides compelling evidence of the significance of the BCRCin driving B-cell malignancies. Additional molecular targets have beenidentified downstream of the BCRC, such as PI3K delta and BCL2, anddrugs blocking the activity of these targets are also shown to havesignificant clinical activity.

As a consequence of the BCR's sequence homology to serum Ig, developingspecific anti-membrane Ig therapy was a hurdle. Specific mIgM targetingin vivo was thought not to be feasible, as the drug or biologic wouldbind to the circulating IgM in blood prior to reaching the cell surfaceB-cell membrane mIgM. A unique set of sequences previously identified inthe membrane-bound Igs, designated proximal domains (PDs), are notexpressed in serum Igs. These PDs are Ig class specific and for mIgMconstitutes a 13 amino acid peptide. However, attempts to produceanti-mIgM PD antibodies were not successful due to the lowimmunogenicity of the PD peptide, its hydrophobicity, and the resultantlow affinity of the generated antibodies. In contrast, efforts toproduce mIgE PD have resulted in several functionally distinct versions.See, e.g., U.S. Pat. No. 8,137,670; U.S. Pat. No. 8,404,236; PoggianellaM, et al., J Immunol. 177:3597-3605 (2006); Feichter S, et al., JImmunol 5499-5505 (2008).

There is a need for antibodies that have a high level of specificity forB-cell mIgM in order to internalize the receptor, inhibit cell growth,induce apoptosis or deliver drugs, toxins or radioisotopes to these mIgMB-cells, while sparing normal lymphocytes (non-mIgM expressing B-cells)and non-lymphatic tissues from toxicity. Such antibodies can also beused in diagnostics of B-cell lymphomas and B-cell leukemias. Theseuniquely specific antibodies will allow for the first time the abilityto separate membrane IgM from serum IgM by immune-affinity methodology.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Cell line CRL 1648 Scanning Immuno-Electron microscopy(Burkitt's lymphoma). FIG. 1A is a micrograph showing the control-IgG2bisotype matched control antibody plus secondary goat anti-mouse Ig-gold.FIGS. 1B and 1C are micrographs showing monoclonal antibody mAb4-2b(hereinafter “mAb 4”) binding to 2 different cells of CRL 1648 at thesame magnification as the control antibody in FIG. 1A. FIG. 1D is amicrograph showing monoclonal antibody mAb 4 binding to a third CRL 1648cell at a higher magnification compared to the control antibody of FIG.1A. Bright white spots represent immune gold particles-goat anti-mouseIg reacting with the mAb 4 monoclonal antibody on the cell surface.

FIGS. 2A-2E. Cell line CRL 1648 Scanning Immuno-Electron microscopy(Burkitt's lymphoma). FIG. 2A is a micrograph showing monoclonalantibody mAb 4 binding to a glutaraldehyde fixed CRL 1648 cell. FIG. 2Bis a micrograph showing micro-clusters of BCRC. FIG. 2C is a micrographshowing that when CRL 1648 cells were incubated with mAb 4 at 37° C. for30 minutes, then fixed and stained with goat-anti-mouse Ig, there was alack of detectable monoclonal antibody mAb 4 on the membrane due to BCRCinternalization. FIG. 2D is a micrograph showing that when CRL 1648cells were incubated with mAb 4 at 37° C. for 15 minutes, then fixed andstained with goat-anti-mouse Ig, residual bound monoclonal antibody mAb4 was seen because internalization was incomplete. FIG. 2E is amicrograph showing that when CRL 1648 cells were incubated withmonoclonal antibody mAb 4 at 37° C. for 30 minutes, then fixed andstained with goat-anti-hu-IgM, BCRC is not detectable.

FIG. 3. Cell line CRL 1596 Scanning Immuno-Electron microscopy(Burkitt's lymphoma). FIG. 3 is a micrograph in which monoclonalantibody mAb 4 binding is represented by gold particles-goat-anti-mouseIg reactivity, demonstrating specific binding to long projections aswell as the cell surface of CRL 1596.

FIGS. 4A-4B. Cell line CRL 2260 Scanning Immuno-Electron microscopy(Diffuse mixed B-cell lymphoma). FIG. 4A is a micrograph providing ahigh magnification view of a dense micro-cluster that shows specificbinding of monoclonal antibody mAb 4, represented by goldparticles-goat-anti-mouse Ig reactivity, to CRL 2260. FIG. 4B is amicrograph providing a topographic view of the same field in FIG. 4Athat shows specific binding of monoclonal antibody mAb 4, represented bygold particles-goat-anti-mouse Ig reactivity, to CRL 2260, Diffuse mixedB-cell Lymphoma.

FIGS. 5A-5B. Cell line CRL 3006 Scanning Immuno-Electron microscopy(Mantle cell lymphoma). FIG. 5A is a micrograph providing a highmagnification view of a dense micro-cluster that shows specific bindingof monoclonal antibody mAb 4, represented by goldparticles-goat-anti-mouse Ig reactivity, in deep clefts of CRL 3006.FIG. 5B is a micrograph providing a topographic view of the same fieldin FIG. 5A that shows specific binding of monoclonal antibody mAb 4,represented by gold particles-goat-anti-mouse Ig reactivity, to CRL3006, Mantle Cell Lymphoma.

FIGS. 6A-6F. FIG. 6A is a graph comparing the effect of monoclonalantibody mAb 4 versus an isotype-matched control antibody on inhibitinggrowth of mIgM-expressing B-cell line CRL 1648 (CRL 1648 Mκ) (Burkitt'slymphoma). FIG. 6B is a graph comparing the effect of monoclonalantibody mAb2-2b (hereinafter “mAb 2”) versus an isotype-matched controlantibody on inhibiting growth of mIgM-expressing B-cell line CRL 1648(CRL 1648 Mκ). FIG. 6C is a graph comparing the effect of monoclonalantibody mAb 4 versus an isotype-matched control antibody on inhibitinggrowth of control B-cell line expressing mIgG, CRL 2632 (CRL 2632 Gκ)(Diffuse large cell lymphoma). FIG. 6D is a graph comparing the effectof monoclonal antibody mAb 4 versus an isotype-matched control antibodyon inhibiting growth of mIgM-expressing B-cell line CRL 2958 (CRL2958Mλ) (Diffuse large cell lymphoma). FIG. 6E is a graph comparing theeffect of monoclonal antibody mAb 4 versus an isotype-matched controlantibody on inhibiting growth of mIgM-expressing B-cell line CRL 1596(CRL 1596 Mλ) (Burkitt's lymphoma). FIG. 6F is a graph comparing theeffect of monoclonal antibody mAb 4 versus an isotype-matched controlantibody on inhibiting growth of mIgM-expressing B-cell line CRL 1432(CRL 1432 Mλ) (Burkitt's lymphoma).

FIG. 7 is a graph showing the effect of monoclonal antibody mAb 4 ongrowth of mIgM-expressing B-cell line CRL 1648 at cell dilutions of 20cells/well, 100 cells/well, 250 cells/well, 500 cells/well and 1,000cells/well.

SUMMARY OF THE INVENTION

The present invention relates to isolated antibodies specificallytargeting membrane bound IgM (mIgM) of the B-cell Receptor Complex inB-cell lymphomas and leukemias. The antibodies of the invention may be arecombinant antibody. The antibodies of the invention may be monoclonalor a class-switched monoclonal derived from a monoclonal antibody of theinvention, and a monoclonal antibody may be a mouse antibody, a humanantibody, a chimeric antibody, or a humanized antibody.

Also included in the present invention are antigen binding regions(CDRs) derived from the light and/or heavy chain variable regions ofsaid antibodies. The antibodies of the invention may be a recombinantantibody. The antibodies of the invention may be monoclonal, and amonoclonal antibody may be a human antibody, a chimeric antibody, or ahumanized antibody.

The present invention includes an antibody that comprises a heavy chainvariable region (VH) encoded by the nucleic acid sequence of SEQ ID NO:1; and/or a light chain variable region (VL) encoded by the nucleic acidsequence of SEQ ID NO: 3.

The present invention includes an antibody that comprises a heavy chainvariable region (VH) having the amino acid sequence depicted in SEQ IDNO: 2 and/or a light chain variable region (VL) having the amino acidsequence depicted in SEQ ID NO: 4.

The present invention includes an antibody that comprises a heavy chainvariable region (VH) comprising a VH CDR1 having the amino acid sequenceof SEQ ID NO: 5; a VH CDR2 having the amino acid sequence of SEQ ID NO:6; and/or a VH CDR3 having the amino acid sequence of SEQ ID NO: 7;and/or a light chain variable region (VL) comprising a VL CDR1 havingthe amino acid sequence of SEQ ID NO: 8; a VL CDR2 having the amino acidsequence of SEQ ID NO: 9; and/or a VL CDR3 having the amino acidsequence of SEQ ID NO: 10.

The present invention includes an antibody that comprises a heavy chainvariable region (VH) comprising a VH CDR1 having the amino acid sequenceof SEQ ID NO: 5; a VH CDR2 having the amino acid sequence of SEQ ID NO:6; and a VH CDR3 having the amino acid sequence of SEQ ID NO: 7; and/ora light chain variable region (VL) comprising a VL CDR1 having the aminoacid sequence of SEQ ID NO: 8; a VL CDR2 having the amino acid sequenceof SEQ ID NO: 9; and a VL CDR3 having the amino acid sequence of SEQ IDNO: 10.

The present invention includes an antibody wherein the VH is encoded bya nucleotide sequence that hybridizes under stringent conditions to thecomplement of a nucleotide sequence that encodes the amino acid sequenceof SEQ ID NO: 2; and/or a VL encoded by a nucleotide sequence thathybridizes under stringent conditions to the complement of a nucleotidesequence that encodes the amino acid sequence of SEQ ID NO: 4.

The present invention includes a VL sequence having at least 95%sequence identity to that set forth in SEQ ID NO: 4, and a VH sequenceat least 95% sequence identity to that set forth in SEQ ID NO: 2.

The present invention includes human antigen-binding antibody fragmentsof the antibodies of the present invention including, but not limitedto, Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), diabodies, triabodies, orminibodies.

The present invention includes a monoclonal antibody designated mAb1-1produced by a hybridoma cell line from fusion 117 (ATCC deposit number)and clones thereof.

The present invention includes a monoclonal antibody designated mAb2-2bproduced by a hybridoma cell line from fusion 118 (ATCC deposit number)and clones thereof.

The present invention includes a monoclonal antibody designated mAb3-2bproduced by a hybridoma cell line from fusion 118 (ATCC deposit number______) and clones thereof.

The present invention includes a monoclonal antibody designated mAb4-2bproduced by hybridoma cell line from fusion 119 (ATCC deposit number______) and clones thereof.

The present invention relates to the antibody or antigen bindingfragment of the present invention decreasing B-cell Receptor activity.

The present invention includes an antibody of the present inventionfurther comprising a label.

The present invention includes the antibodies targeting the mIgM in theB-cell Receptor Complex in B-cell lymphomas and leukemias describedabove further comprising a cytotoxin, radioisotope or immunotoxin andtheir use in treating B-cell lymphomas and leukemias.

The present invention also includes antibodies that bind the sameepitopes as antibody mAb4-2b.

The present invention also includes antibodies that bind the sameepitope as antibody mAb1-1, mAb2-2b or mAb3-2b, including thoseantibodies that bind all isomeric forms of membrane proximal domain.

The present invention includes a composition comprising an antibody ofthe present invention and at least one of a physiologically acceptablecarrier, diluent, excipient, or stabilizer.

The present invention relates to the use of an antibody or antigenbinding fragment of the invention for the preparation of a medicament totreat B-cell lymphomas and leukemias in a subject.

The present invention relates to the use of the antibody or antigenbinding fragment of the invention for the preparation of a medicament todecrease the activity of B-cell Receptor Complex.

The present invention includes a method of ameliorating or treating aB-cell lymphoma or leukemia in a patient, comprising administering tothe patient an effective amount of an antibody that binds to B-cell mIgMand induces cell growth inhibition and/or apoptosis.

The present invention includes a composition comprising the antibodiesaccording to the present invention in combination with a physiologicallyacceptable carrier, diluents, excipient, or stabilizer.

The present invention includes a method of killing or inhibiting thegrowth of B cells in a subject, comprising administering an effectiveamount of an antibody according to the present invention to a subject inneed thereof, thereby killing or inhibiting the growth of the B cells ina subject.

The present invention includes a method of killing or inhibiting thegrowth of B cells in a subject, comprising administering an effectiveamount of the antibody of claim 1 to a subject in need thereof incombination with one or more anti-B-cell antibodies, a cytotoxin, and/ora radioisotope, thereby killing or inhibiting the growth of the B cellsin a subject. The present invention includes a hybridoma that producesan antibody of the present invention.

The present invention includes a hybridoma cell line designated ATCC forproducing the monoclonal antibody designated mAb1-1.

The present invention includes a hybridoma cell line designated ATCC forproducing the monoclonal antibody designated mAb2-2b.

The present invention includes a hybridoma cell line designated ATCC forproducing the monoclonal antibody designated mAb3-2b.

The present invention includes a hybridoma cell line designated ATCC forproducing the monoclonal antibody designated mAb4-2b.

The present invention relates to a complex comprising B-cell membraneIgM and any one of the antibody or antigen binding fragments describedherein.

The present invention includes a method of producing an antibodycomprising culturing a hybridoma cell line of the present inventionunder conditions suitable for the production of the antibody, andisolation of the antibody.

The present invention relates to an isolated nucleic acid encoding anyof the antibodies or antigen binding fragments of the invention.

The present invention includes an isolated nucleic acid moleculeencoding an antibody of the present invention wherein the nucleotidesequence comprises SEQ ID NO: 1 and/or 3.

The present invention includes an isolated nucleic acid moleculeencoding an antibody of the present invention comprising the amino acidsequence of SEQ ID NO: 2 or 4.

The present invention includes an isolated nucleic acid moleculecomprising a nucleic acid sequence that encodes a heavy chain variableregion (VH) amino acid sequence set forth in SEQ ID NO: 2, and/or alight chain variable region (VL) amino acid sequence set forth in SEQ IDNO: 4.

The present invention relates to an expression vector comprising anisolated nucleic acid encoding any of the antibodies or antigen bindingfragments of the invention. In one embodiment, the isolated nucleic acidencodes any of the VH or VL chains described herein. The invention alsorelates to a host cell comprising any of the expression vectorsdescribed herein.

The present invention relates to isolated polypeptides comprising the VHor VL domains or any of the antibodies or antigen binding fragments ofthe invention.

In certain embodiments, these nucleic acids, expression vectors orpolypeptides of the invention are useful in methods of making anantibody.

The present invention includes a method for the detection of B-cell mIgMin a sample, comprising contacting the sample with an antibody of thepresent invention.

The present invention includes a method for the detection of B-cell mIgMin a patient sample, including determining minimal residual disease,comprising contacting the sample with an antibody of the presentinvention.

The present invention includes a method for the detection of B-cell mIgMmicro-clustering thereby allowing for sub-typing B-cell malignancies andproviding patient-specific prognostic information in a sample,comprising contacting the sample with an antibody of the presentinvention.

The present invention includes a method for purifying B-cell Receptorsusing an antibody of the present invention.

The present invention includes a kit comprising an antibody of thepresent invention in a predetermined amount in a container, and a bufferin a separate container.

The present invention includes a kit comprising a composition of thepresent invention described above in a predetermined amount in acontainer, and a buffer in a separate container.

DETAILED DESCRIPTION OF THE INVENTION

This invention is not limited to the particular methodology, protocols,cell lines, or reagents described herein because they may vary. Further,the terminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention. As used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise, e.g., reference to “a host cell” includes aplurality of such host cells. Unless defined otherwise, all technicaland scientific terms and any acronyms used herein have the same meaningsas commonly understood by one of ordinary skill in the art in the fieldof the invention. Although any methods and materials similar orequivalent to those described herein can be used in the practice of thepresent invention, the exemplary methods, devices, and materials aredescribed herein.

Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   -   ADCC Antibody-dependent cellular cytotoxicity    -   ATCC American Type Culture Collection    -   BCL2 or Bcl-2 B-cell lymphoma 2    -   BCR B-cell Receptor    -   BCRC B-cell Receptor Complex    -   BTK Tyrosine-protein kinase BTK    -   CDC Complement-dependent cytotoxicity    -   CDR Complementarity determining region in the immunoglobulin        variable regions, defined using the Kabat numbering system    -   CHO Chinese hamster ovary    -   CLL Chronic lymphocytic leukemia    -   ELISA Enzyme-linked immunosorbant assay    -   FM Fluorescent microscopy    -   FR Antibody framework region: the immunoglobulin variable        regions excluding the CDR regions    -   HRP Horseradish peroxidase    -   IFN Interferon    -   IC50 Concentration resulting in 50% inhibition    -   Ig Immunoglobulin    -   IgA Immunoglobulin A    -   IgD Immunoglobulin D    -   IgE Immunoglobulin D    -   IgG Immunoglobulin G    -   IgM Immunoglobulin M    -   Kabat An immunoglobulin alignment and numbering system pioneered        by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological        Interest, 5th Ed. Public Health Service, National Institutes of        Health, Bethesda, Md.)    -   mAb, Mab, or MAb Monoclonal antibody    -   mAb 1 or mAb1 Monoclonal antibody mAb1-1    -   mAb 2 or mAb2 Monoclonal antibody mAb2-2b    -   mAb 3 or mAb3 Monoclonal antibody mAb3-2b    -   mAb 4 or mAb4 Monoclonal antibody mAb4-2b    -   mIg Cell-surface membrane bound immunoglobulin    -   mIgA Cell-surface membrane bound immunoglobulin A    -   mIgD Cell-surface membrane bound immunoglobulin D    -   mIgE Cell-surface membrane bound immunoglobulin D    -   mIgG Cell-surface membrane bound immunoglobulin G    -   mIgM Cell-surface membrane bound immunoglobulin M    -   PCR Polymerase chain reaction    -   PD Proximal domains    -   PI3K Phosphoinositide 3-kinase    -   PK Pharmacokinetics    -   SEM Scanning Immuno-Electron microscopy    -   V region The segment of IgG chains which is variable in sequence        between different antibodies. It extends to Kabat residue 109 in        the light chain and 113 in the heavy chain.    -   VH Immunoglobulin heavy chain variable region    -   VK Immunoglobulin kappa light chain variable region    -   VL Immunoglobulin light chain variable region

DEFINITIONS

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,applicants desire that the following terms be given the particulardefinition as defined below.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, or 80%, or 90%, or 95% sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95%, or 97% sequence identity to the reference nucleicacid sequence.

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The term “antibody” is used in the broadest sense, includingimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end. Moreover, the term “antibody” (Ab) or “monoclonalantibody” (mAb) is meant to include both intact molecules, as well asantibody fragments (such as, for example, Fab and F(ab′)₂ fragments)that are capable of specifically binding to a protein. Fab and F(ab′)₂fragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl, et al., JNucl Med 24:316 (1983)).

As used herein, “anti-B-cell mIgM antibody” means an antibody whichbinds to human B-cell mIgM in such a manner so as to inhibit cellgrowth, internalize mIgM or induce apoptosis of the B-cells having thismIgM epitope.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular target.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions both in the light chain and the heavy chain variable domains.The more highly conserved portions of variable domains are called theframework (FR). As is known in the art, the amino acid position/boundarydelineating a hypervariable region of an antibody can vary, depending onthe context and the various definitions known in the art. Some positionswithin a variable domain may be viewed as hybrid hypervariable positionsin that these positions can be deemed to be within a hypervariableregion under one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions. Theinvention provides antibodies comprising modifications in these hybridhypervariable positions. The variable domains of native heavy and lightchains each comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of the targetbinding site of antibodies (see Kabat, et al. Sequences of Proteins ofImmunological Interest, National Institute of Health, Bethesda, Md.(1987)). As used herein, numbering of immunoglobulin amino acid residuesis done according to the immunoglobulin amino acid residue numberingsystem of Kabat, et al., unless otherwise indicated.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Thephrase “antigen binding fragment” of an antibody is a compound havingqualitative biological activity in common with a full-length antibody.For example, an antigen binding fragment of an anti-B-cell mIgM antibodyis one which can bind to a B-cell mIgM receptor in such a manner so asto prevent or substantially reduce the ability of such molecule fromhaving the ability to bind to the B-cell mIgM. As used herein,“functional fragment” with respect to antibodies, refers to Fv, F(ab)and F(ab′)₂ fragments. An “Fv” fragment is the minimum antibody fragmentwhich contains a complete target recognition and binding site. Thisregion consists of a dimer of one heavy and one light chain variabledomain in a tight, non-covalent association (V_(H)—V_(L) dimer). It isin this configuration that the three CDRs of each variable domaininteract to define a target binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer target bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for a target) has theability to recognize and bind target, although at a lower affinity thanthe entire binding site. “Single-chain Fv” or “scFv” antibody fragmentscomprise the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the scFv to form the desired structure fortarget binding.

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts and the naturally present class-switchvariants containing identical CDR sequences. Monoclonal antibodies arehighly specific, being directed against a single target site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the target. In addition totheir specificity, monoclonal antibodies are advantageous in that theymay be synthesized by the hybridoma culture, uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies for use with the present invention may be isolatedfrom phage antibody libraries using the well-known techniques. Theparent monoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohler,et al., Nature 256:495 (1975), or may be made by recombinant methods.While monoclonal antibodies are usually produced in mice with identicalgenetic background as the fusion multiple myeloma partner (e.g., SP2/0),previously Yin et al., J Immunol Methods 144:165-173 (1991), havereported the use of non-identical partners in order to take advantage ofgenetically enhanced immune reactivity and affinity in different mousestrains.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from non-human immunoglobulins, such as rator mouse antibody, and human immunoglobulins constant regions, typicallychosen from a human immunoglobulin template. More recently, chimericstructures comprising the binding variable sequences of the monoclonalantibody and cell receptors have been developed. Chimeric shall alsorefer to antibodies having the humanized variable region sequences andmouse constant region sequences to allow for routineimmunohistochemistry, flow cytometry or other assays optimized formurine reagents.

“Humanized” forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibody mayalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin template chosen.

As used herein, “human antibodies” include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati, et al.

The terms “cell,” “cell line,” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included. The “host cells” used inthe present invention generally are prokaryotic or eukaryotic hosts.

“Transformation” of a cellular organism with DNA means introducing DNAinto an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. “Transfection” of acellular organism with DNA refers to the taking up of DNA, e.g., anexpression vector, by the cell or organism whether or not any codingsequences are in fact expressed. The terms “transfected host cell” and“transformed” refer to a cell in which DNA was introduced. The cell istermed “host cell” and it may be either prokaryotic or eukaryotic.Typical prokaryotic host cells include various strains of E. coli.Typical eukaryotic host cells are mammalian, such as Chinese hamsterovary or cells of human origin. The introduced DNA sequence may be fromthe same species as the host cell of a different species from the hostcell, or it may be a hybrid DNA sequence, containing some foreign andsome homologous DNA.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA in a suitable host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control the terminationof transcription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. In the present specification, “plasmid” and “vector” aresometimes used interchangeably, as the plasmid is the most commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent function as and which are, orbecome, known in the art. “Mammal” for purposes of treatment refers toany animal classified as a mammal, including human, domestic and farmanimals, nonhuman primates, and zoo, sports, or pet animals, such asdogs, horses, cats, cows, etc.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to a moleculeor protein, e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column).

The terms “activation,” “stimulation,” and “treatment,” as it applies tocells or to receptors, may have the same meaning, e.g., activation,stimulation, or treatment of a cell or receptor with a ligand, unlessindicated otherwise by the context or explicitly. “Ligand” encompassesnatural and synthetic ligands, e.g., cytokines, cytokine variants,analogues, muteins, and binding compounds derived from antibodies.“Ligand” also encompasses small molecules, e.g., peptide mimetics ofcytokines and peptide mimetics of antibodies. “Activation” can refer tocell activation as regulated by internal mechanisms as well as byexternal or environmental factors. “Response,” e.g., of a cell, tissue,organ, or organism, encompasses a change in biochemical or physiologicalbehavior, e.g., concentration, density, adhesion, or migration within abiological compartment, rate of gene expression, or state ofdifferentiation, where the change is correlated with activation,stimulation, or treatment, or with internal mechanisms such as geneticprogramming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., immunological activity/mg protein, concentration in abiological compartment, or the like. “Activity” may refer to modulationof components of the innate or the adaptive immune systems.

The term “proliferative activity” encompasses an activity that promotes,that is necessary for, or that is specifically associated with, e.g.,normal cell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

The terms “administration” and “treatment,” as it applies to an animal,human, experimental subject, cell, tissue, organ, or biological fluid,refers to contact of an exogenous pharmaceutical, therapeutic,diagnostic agent, or composition to the animal, human, subject, cell,tissue, organ, or biological fluid. “Administration” and “treatment” canrefer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell. The term“subject” includes any organism, preferably an animal, more preferably amammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.

The terms “treat” or “treating” means to administer a therapeutic agent,such as a composition containing any of the antibodies or antigenbinding fragments of the present invention, internally or externally toa subject or patient having one or more disease symptoms, or beingsuspected of having a disease or being at elevated at risk of acquiringa disease, for which the agent has therapeutic activity. Typically, theagent is administered in an amount effective to alleviate one or moredisease symptoms in the treated subject or population, whether byinducing the regression of or inhibiting the progression of suchsymptom(s) by any clinically measurable degree. The amount of atherapeutic agent that is effective to alleviate any particular diseasesymptom (also referred to as the “therapeutically effective amount”) mayvary according to factors such as the disease state, age, and weight ofthe patient, and the ability of the drug to elicit a desired response inthe subject. Whether a disease symptom has been alleviated can beassessed by any clinical measurement typically used by physicians orother skilled healthcare providers to assess the severity or progressionstatus of that symptom. While an embodiment of the present invention(e.g., a treatment method or article of manufacture) may not beeffective in alleviating the target disease symptom(s) in every subject,it should alleviate the target disease symptom(s) in a statisticallysignificant number of subjects as determined by any statistical testknown in the art such as the Student's t-test, the chi² test, the U testaccording to Mann and Whitney, the Kruskal-Wallis test (H test),Jonckheere-Terpstra test and the Wilcoxon signed-rank test. The term“treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of an agonist or antagonist to a human or animalsubject, a cell, tissue, physiological compartment, or physiologicalfluid. “Treatment of a cell” also encompasses situations where theagonist or antagonist contacts the receptor, e.g., in the fluid phase orcolloidal phase, but also situations where the agonist or antagonistdoes not contact the cell or the receptor.

Cell Lines

The following eleven human B-cell lineage cell lines and one murine cellline are referred to throughout the detailed description and examples ofthe invention as follows:

1. CRL 1432—Namalwa mIgM-L Burkitt's

2. CRL 1596—Ramos sIgM mIgM-L Burkitt's

3. CRL 1647—ST 486 sIgM mIgM-K Burkitt's

4. CRL 1648—CA 46 mIgM-K Burkitt's

5. CRL 1649—MC 116 mIgM-L Undifferentiated lymphoma

6. CRL 2260—HT mIgM-K Diffuse mixed B-cell lymphoma

7. CRL 2289—DB mIgG-L Large B-cell Lymphoma

8. CRL 2568—H2.8 murine IgG1-K Myeloma

9. CRL 2632—Diffuse large cell lymphoma IgGk

10. CRL 2958—SU-DHL-5 Diffuse large cell lymphoma mIgM

11. CRL 3006—JeKo-1-L mantle cell Lymphoma mIgM

12. SK007—Lymphoma mIgE

These cell lines were obtained from the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110, and tested forPD expression by RT-PCR. Cytoplasmic and secreted IgM fractions wereconfirmed by ELISA of supernatants and washed 0.01% NP-40 cell lysates.

Antibody Generation

The antibodies of the present invention may be generated by any suitablemethod known in the art. The antibodies of the present invention maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan (Harlow, et al., Antibodies:a Laboratory Manual, Cold spring Harbor Laboratory Press, 2nd ed.(1988)), which is hereby incorporated herein by reference in itsentirety).

For example, an immunogen as described above may be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc., to induce the production of sera containing polyclonal antibodiesspecific for the antigen. The selection of the specific mouse strain forimmunization may be critical for immunogens that elicit poor responses.

Mouse strain tolerance may be overcome by pre-screening and selectingstrains, such as testing those with autoimmune defects or more wild typeimmune reactivity. The administration of the immunogen may entail one ormore injections of an immunizing agent and, if desired, an adjuvant.Various adjuvants may be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, Multiple antigen peptide, and potentiallyuseful human adjuvants such as BCG (Bacille Calmette-Guerin) andCorynebacterium parvum. Additional examples of adjuvants which may beemployed include keyhole limpet hemocyanin with bound immunogen peptide,multiple antigen polypeptide with bound immunogen peptide, and theMPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalosedicorynomycolate). Immunization protocols are well known in the art andmay be performed by any method that elicits an immune response in theanimal host chosen. Adjuvants are also well known in the art.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or through IV. The immunogen may include a targetpeptide, for membrane IgM: EGEVSADEEGFEN (SEQ ID NO: 11) or for membraneIgG: ELQLEESCAEAQDGELDG (SEQ ID NO: 12), purified B-cell mIgM, a fusionprotein, or variants thereof. The target peptide for membrane IgM,EGEVSADEEGFEN (SEQ ID NO: 11), has 100% homology to a human IgM peptidehCG2038942 (Accession No. EAW81938.1) that has the sequence EGEVSEDEEGFE(SEQ ID NO: 13). Depending upon the nature of the peptides (i.e.,percent hydrophobicity, percent hydrophilicity, stability, net charge,isoelectric point, multiple isomeric forms, etc.), it may be useful toconjugate the immunogen to a protein known to be immunogenic in themammal being immunized. Such conjugation includes either chemicalconjugation by active derivation of chemical functional groups to boththe immunogen and the immunogenic protein to be conjugated such that acovalent bond is formed, or through fusion-protein based methodology, orother methods known to the skilled artisan. Examples of such immunogenicproteins include, but are not limited to, keyhole limpet hemocyanin,multiple antigen peptide, ovalbumin, serum albumin, bovinethyroglobulin, soybean trypsin inhibitor, and promiscuous T helperpeptides. Various adjuvants may be used to increase the immunologicalresponse as described above.

The antibodies of the present invention may comprise monoclonalantibodies.

Monoclonal antibodies are antibodies which recognize a single antigenicsite. Their uniform specificity makes monoclonal antibodies much moreuseful than polyclonal antibodies, which usually contain antibodies thatrecognize a variety of different antigenic sites. Monoclonal antibodiesmay be prepared using hybridoma technology, such as those described byKohler, et al., Nature 256:495 (1975); U.S. Pat. No. 4,376,110; Harlow,et al. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2nd ed. (1988) and Hammerling, et al., Monoclonal Antibodies andT-Cell Hybridomas, Elsevier (1981), recombinant DNA methods, or othermethods known to the artisan. Other examples of methods which may beemployed for producing monoclonal antibodies include, but are notlimited to, the human B-cell hybridoma technique (Kosbor, et al.,Immunology Today 4:72 (1983); Cole, et al., Proc Natl Sci USA 80:2026(1983)), and the EBV-hybridoma technique (Cole, et al., MonoclonalAntibodies and Cancer Therapy, pp. 77-96. Alan R. Liss (1985)). Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,and IgA, IgD and any subclass thereof. The hybridoma producing the mAbof this invention may be cultivated in vitro or in vivo.

In the hybridoma model, a host such as a mouse, a humanized mouse, amouse with a human immune system, hamster, rabbit, camel, or any otherappropriate host animal, is immunized to elicit lymphocytes that produceor are capable of producing antibodies that will specifically bind tothe protein used for immunization. In the present invention, a mouseutilizing a human Ig genetic repertoire could not be used, as theinitial B-cell itself would be a target of the desired antibody andwould result in apoptosis of the B-cell. Alternatively, lymphocytes maybe immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, pp. 59-103 (1986)).

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Typically,a rat or mouse myeloma cell line is employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), substances that prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as FIATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from the MOPC-21 and MPC-11 mouse tumors and SP2/0 orX63-Ag8-653 cells available from the ATCC, 10801 University Boulevard,Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J Immunol 133:3001 (1984); Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc. pp. 51-63 (1987)). The mouse myeloma cell line NSO may alsobe used (European Collection of Cell Cultures, Salisbury, Wilshire, UK).

The culture medium in which hybridoma cells are grown is assayed forproduction of monoclonal antibodies directed against peptides, e.g., forIgM, EGEVSADEEGFEN (SEQ ID NO: 11) and for IgG, ELQLEESCAEAQDGELDG (SEQID NO: 12). The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA), immune blot,Westerns or enzyme-linked immunoabsorbent assay (ELISA). Such techniquesare known in the art and within the skill of the artisan. The bindingaffinity of the monoclonal antibody can, for example, be determined by aScatchard analysis (Munson, et al., Anal Biochem 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium.In addition, the hybridoma cells may be grown in vivo as ascites tumorsin an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated or isolated from the culture medium, ascites fluid, or serumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelexclusion chromatography, gel electrophoresis, dialysis, or affinitychromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hybridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. The hybridoma cells serve as a source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, NSO cells, Simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc Natl Acad SciUSA 81:6851 (1984)) or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibody or the present invention or antigen binding fragmentthereof that binds to membrane bound IgM (mIgM) of B-cell ReceptorComplex (BCRC) can comprise one, two, three, four, five, or six of thecomplementarity determining regions (CDRs) of the antibodies disclosedherein. The one, two, three, four, five, or six CDRs may beindependently selected from the CDR sequences of a single describedantibody of the invention (e.g., Table 1, Table 2). In certainembodiments, one two or three CDRs are selected from the VL CDRs (e.g.,Table 1; SEQ ID NOs:8-10) of the described antibody and/or one, two orthree CDRs selected from the VH CDRs (e.g., Table 2; SEQ ID NOs: 5-7) ofthe described invention.

The isolated antibody of the present invention or antigen-bindingfragment thereof that binds to mIgM of BCRC comprises an antibody lightchain variable (VL) domain comprising one or more of CDR-L1, CDR-L2 orCDR-L3 of antibody mAb4-2b.

The isolated antibody of the present invention or antigen-bindingfragment thereof that binds to mIgM of BCRC comprises an antibody heavychain variable (VH) domain comprising one or more of CDR-H1, CDR-H2 orCDR-H3 of antibody mAb4-2b.

In a further embodiment the isolated antibody or antigen-bindingfragment thereof that binds to mIgM of BCRC comprises an antibody lightchain variable (VL) domain comprising one or more of CDR-L1, CDR-L2 orCDR-L3 of antibody mAb4-2b, and an antibody heavy chain variable (VH)domain comprising one or more of CDR-H1, CDR-H2 or CDR-H3 of antibodymAb4-2b. Sequences of light and heavy chain CDRs of the mAb4-2b antibodyof the present invention are provided in Tables 1 and 2, respectively.

TABLE 1 Light Chain CDRs Antibody CDR1 CDR2 CDR3 mAb4-2b SEQ ID NO: 8SEQ ID NO: 9 SEQ ID NO: 10

TABLE 2 Heavy Chain CDRs Antibody CDR1 CDR2 CDR3 mAb4-2b SEQ ID NO: 5SEQ ID NO: 6 SEQ ID NO: 7

The invention also provides isolated polypeptides comprising the VLdomains (e.g., SEQ ID NO: 4) and isolated polypeptides comprising the VHdomains (e.g., SEQ ID NO: 2) of the antibodies of the invention. Inother embodiments the invention provides antibodies or antigen bindingfragment thereof that specifically binds to mIgM of BCRC and has VLdomains and VH domains with at least 50%, 75%, 80%, 85%, 90%, 95%, 98%or 99% sequence identity with SEQ ID NOs: 2 and 4 while still exhibitingthe desired binding and functional properties. In another embodiment,the antibody or antigen binding fragment of the present inventioncomprises VL and VH domains (with and without signal sequence) having upto 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative ornon-conservative amino acid substitutions, while still exhibiting thedesired binding and functional properties.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g., charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Various embodiments of theantibody or antigen binding fragment of the present invention comprisepolypeptide chains with the sequences disclosed herein, e.g., SEQ IDNOs: 2, 4, 5, 6, 7, 8, 9, and 10, or polypeptide chains comprising up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative aminoacid substitutions. Exemplary conservative substitutions are set forthin Table 3.

TABLE 3 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention arealso contemplated by the present invention. “Function-conservativevariants,” as used herein, refers to antibodies or fragments in whichone or more amino acid residues have been changed without altering adesired property, such as antigen affinity and/or specificity. Suchvariants include, but are not limited to, replacement of an amino acidwith one having similar properties, such as the conservative amino acidsubstitutions of Table 3.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that specifically binds human mIgM of BCRC andhas VL domains or VH domains with at least 95%, 90%, 85%, 80%, 75% or50% sequence homology to one or more of the VL domains or VH domainsdescribed herein, and exhibits specific binding to human mIgM of BCRC.In another embodiment, the binding antibody or antigen binding fragmentthereof of the present invention comprises VL and VH domains (with andwithout signal sequence) having up to 1, 2, 3, 4, or 5 or more aminoacid substitutions, and exhibits specific binding to human mIgM of BCRC.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto, et al.,J Biochem Biophys Methods 24:107 (1992); Brennan, et al., Science 229:81(1985)). For example, Fab and F(ab′)₂ fragments of the invention may beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). F(ab′)₂ fragments contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain.However, these fragments can now be produced directly by recombinanthost cells. For example, the antibody fragments can be isolated from anantibody phage library. Alternatively, F(ab′)₂-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′),fragments (Carter, et al., Bio/Technology 10:163 (1992). According toanother approach, F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner. Inother embodiments, the antibody of choice is a single chain Fv fragment(Fv) (PCT Publication No. WO 93/16185).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques 4:214(1986); Gillies, et al., J Immunol Methods 125:191 (1989); U.S. Pat.Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated hereinby reference in their entirety.

A humanized antibody is designed to have greater homology to a humanimmunoglobulin than animal-derived monoclonal antibodies. Humanizationis a technique for making a chimeric antibody wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. Humanized antibodiesare antibody molecules generated in a non-human species that bind thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework (FR) regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., U.S. Pat. No. 5,585,089; Riechmann,et al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties. Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (European App. No. EP 239,400; PCT Publication No. WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (European App. No. EP 592,106; European App. No. EP519,596; Padlan, Molecular Immunology 28:489 (1991); Studnicka, et al.,Protein Engineering 7:805 (1994); Roguska, et al., Proc Natl Acad SciUSA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the methods of Winter and co-workers(Jones, et al., Nature 321:522 (1986); Riechmann, et al., Nature 332:323(1988); Verhoeyen, et al., Science 239:1534 (1988)), by substitutingnon-human CDRs or CDR sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and some possibleFR residues are substituted from analogous sites in rodent antibodies.

It is further important that humanized antibodies retain higher affinityfor the antigen and other favorable biological properties. To achievethis goal, according to a preferred method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of certain residues in the functioning ofthe candidate immunoglobulin sequence, i.e., the analysis of residuesthat influence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), ismaximized, although it is the CDR residues that directly and mostsubstantially influence antigen binding.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.According to the so-called “best-fit” method, the sequence of thevariable domain of a non-human antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of that of the non-human parent antibody isthen accepted as the human FR for the humanized antibody (Sims, et al.,J Immunol 151:2296 (1993); Chothia, et al., J Mol Biol 196:901 (1987)).

Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter, et al., Proc Natl Acad Sci USA 89:4285(1992); Presta, et al., J Immunol 151:2623 (1993)). An antibody of theinvention can comprise any suitable human or human consensus light orheavy chain framework sequences, provided that the antibody exhibits thedesired biological characteristics (e.g., a desired binding affinity).In some embodiments, one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, ormore) additional modifications are present within the human and/or humanconsensus non-hypervariable region sequences. In one embodiment, anantibody of the invention comprises at least a portion (or all) of theframework sequence of human light chain. In one embodiment, an antibodyof the invention comprises at least a portion (or all) of the frameworksequence of human heavy chain. In one embodiment, an antibody of theinvention comprises at least a portion (or all) of human subgroup Iframework consensus sequence. In some embodiments, antibodies of theinvention comprise a human subgroup III heavy chain framework consensussequence. In one embodiment, the framework consensus sequence of theantibody of the invention comprises substitution at position 71, 73and/or 78. In some embodiments of these antibodies, position 71 is A, 73is T and/or 78 is A.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTPublication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole, et al. andBoerner, et al. are also available for the preparation of humanmonoclonal antibodies (Cole, et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss (1985); and Boerner, et al., J Immunol 147:86(1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. See, e.g., Jakobovitis, etal., Proc Acad Sci USA 90:2551 (1993); Jakobovitis, et al., Nature362:255 (1993); Bruggermann, et al., Year in Immunol 7:33 (1993);Duchosal, et al., Nature 355:258 (1992)).

The transgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a polypeptide of the invention.Monoclonal antibodies directed against the antigen can be obtained fromthe immunized, transgenic mice using conventional hybridoma technology.The human immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg, et al., Int Rev Immunol 13:65-93 (1995). For adetailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g. PCT Publication Nos. WO 98/24893; WO 92/01047; WO96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporatedby reference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), andMedarex, Inc. (Princeton, N.J.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Human mAbs could also be made by immunizing mice transplanted with humanperipheral blood leukocytes, splenocytes or bone marrows (e.g., Triomatechniques of XTL). Completely human antibodies which recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach, a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope (Jespers, et al.,Bio/Technology 12:899 (1988)). Of note, human B-cells cannot be used togenerate the specific monoclonal antibodies required, as the monoclonalantibodies would be self-reactive with the primary immunized B-cellinitiating the response.

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art (See, e.g., Greenspan, et al., FASEB J 7:437 (1989);Nissinoff, J Immunol 147:2429 (1991)). For example, antibodies whichbind to and competitively inhibit polypeptide multimerization and/orbinding of a polypeptide of the invention to a ligand can be used togenerate anti-idiotypes that “mimic” the polypeptide multimerizationand/or binding domain and, as a consequence, bind to and neutralizepolypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens toneutralize polypeptide ligand. For example, such anti-idiotypicantibodies can be used to bind a polypeptide of the invention and/or tobind its ligands/receptors, and thereby block its biological activity.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards B-cell mIgM, the other may be for any other antigen,and preferably for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy-chain/light-chain pairs, wherethe two heavy chains have different specificities (Milstein, et al.,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in PCT Publication No. WO93/08829 and in Traunecker, et al., EMBO J 10:3655 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It may have the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transformed into a suitable host organism. For further details ofgenerating bispecific antibodies see, for example Suresh, et al., MethIn Enzym 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single-domain antibodies to B-cell mIgM.Examples of this technology have been described in PCT Publication No.WO9425591 for antibodies derived from Camelidae heavy chain Ig, as wellin US Publication No. 20030130496 describing the isolation of singledomain fully human antibodies from phage libraries.

One can also create a single peptide chain binding molecules in whichthe heavy and light chain Fv regions are connected. Single chainantibodies (“scFv”) and the method of their construction are describedin U.S. Pat. No. 4,946,778. Alternatively, Fab can be constructed andexpressed by similar means. All of the wholly and partially humanantibodies are less immunogenic than wholly murine mAbs, and thefragments and single chain antibodies are also less immunogenic.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty, etal., Nature 348:552 (1990); Clarkson, et al., Nature 352:624 (1991) andMarks, et al., J Biol 222:581 (1991), which describe the isolation ofmurine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity (nMrange) human antibodies by chain shuffling (Marks, et al.,Bio/Technology 10:779 (1992)), as well as combinatorial infection and invivo recombination as a strategy for constructing very large phagelibraries (Waterhouse, et al., Nuc Acids Res 21:2265 (1993)). Thus,these techniques are viable alternatives to traditional monoclonalantibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc Natl Acad Sci USA 81:6851 (1984)).

Another alternative is to use electrical fusion rather than chemicalfusion to form hybridomas. This technique is well established. Insteadof fusion, one can also transform a B cell to make it immortal using,for example, an Epstein Barr Virus, or a transforming gene. See, e.g.,“Continuously Proliferating Human Cell Lines Synthesizing Antibody ofPredetermined Specificity,” Zurawaki, et al., in Monoclonal Antibodies,ed. by Kennett, et al., Plenum Press, pp. 19-33. (1980)). Anti-B-cellmIgM mAbs can be raised by immunizing rodents (e.g., mice, rats,hamsters, and guinea pigs) with B-cell mIgM protein, fusion protein, orits fragments expressed by either eukaryotic or prokaryotic systems.Other animals can be used for immunization, e.g., non-human primates,transgenic mice expression immunoglobulins, and severe combinedimmunodeficient (SCID) mice transplanted with human B lymphocytes.Hybridomas can be generated by conventional procedures by fusing Blymphocytes from the immunized animals with myeloma cells (e.g., Sp2/0and NSO), as described earlier (Kohler, et al., Nature 256:495 (1975)).In addition, anti-B-cell mIgM antibodies can be generated by screeningof recombinant single-chain Fv or Fab libraries from human B lymphocytesin phage-display systems. The specificity of the mAbs to B-cell mIgM canbe tested by ELISA, Western immunoblotting, or other immunochemicaltechniques. The inhibitory activity of the antibodies on CD4+ T cellactivation can be assessed by proliferation, cytokine release, andapoptosis assays. The hybridomas in the positive wells are cloned bylimiting dilution. The antibodies are purified for characterization forspecificity to human B-cell mIgM by the assays described above.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides or nucleic acids, e.g.,DNA, comprising a nucleotide sequence encoding an antibody of theinvention and fragments thereof. Exemplary polynucleotides include thoseencoding antibody chains comprising one or more of the amino acidsequences are described in the Sequence Listing (e.g., SEQ ID NOs: 1 and3). The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions topolynucleotides that encode an antibody of the present invention.

Preferably, the nucleic acids hybridize under low, moderate or highstringency conditions, and encode antibodies that maintain the abilityto specifically bind to mIgM of BCRC. A first nucleic acid molecule is“hybridizable” to a second nucleic acid molecule when a single strandedform of the first nucleic acid molecule can anneal to the second nucleicacid molecule under the appropriate conditions of temperature andsolution ionic strength (see Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, 2^(nd) ed. (1990),3^(rd) ed. (2001)). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Typical low stringencyhybridization conditions include 55° C., 5×SSC, 0.1% SDS and noformamide; or 30% formamide, 5×SSC, 0.5% SDS at 42° C. Typical moderatestringency hybridization conditions are 40% formamide, with 5× or 6×SSCand 0.1% SDS at 42° C. High stringency hybridization conditions are 50%formamide, 5× or 6×SSC at 42° C. or, optionally, at a higher temperature(e.g., 57° C., 59° C., 60° C., 62° C., 63° C., 65° C. or 68° C.). Ingeneral, SSC is 0.15M NaCl and 0.015M Na-citrate. Hybridization requiresthat the two nucleic acids contain complementary sequences, although,depending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the higherthe stringency under which the nucleic acids may hybridize. For hybridsof greater than 100 nucleotides in length, equations for calculating themelting temperature have been derived (see Sambrook, et al., MolecularCloning, A Laboratory Manual, 9.50-9.51, Cold Spring Harbor Laboratory,2^(nd) ed. (1990), 3^(rd) ed. (2001)). For hybridization with shorternucleic acids, e.g., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook, et al., Molecular Cloning, A LaboratoryManual, 11.7-11.8, Cold Spring Harbor Laboratory, 2^(nd) ed. (1990),3^(rd) ed. (2001)).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier, et al., Bio/Techniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory 2^(nd) ed. (1990), 3^(rd) ed. (2001); Ausubel, et al.,eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for example,to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the CDRs by well-known methods, e.g. by comparison to known aminoacid sequences of other heavy and light chain variable regions todetermine the regions of sequence hypervariability. Using routinerecombinant DNA techniques, one or more of the CDRs may be insertedwithin framework regions, e.g., into human framework regions to humanizea non-human antibody, as described supra. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia, et al., J Mol Biol 278: 457(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc Natl Acad Sci 81:851 (1984);Neuberger, et al., Nature 312:604 (1984); Takeda, et al., Nature 314:452(1985)) by splicing genes from a mouse antibody molecule of appropriateantigen specificity together with genes from a human antibody moleculeof appropriate biological activity can be used. As described supra, achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region,e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988);Huston, et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al.,Nature 334:544 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra, et al.,Science 242:1038 (1988)).

Vectors And Host Cells

In another aspect, the present invention provides isolated nucleic acidsequences encoding an antibody as disclosed herein, vector constructscomprising a nucleotide sequence encoding the antibodies of the presentinvention, host cells comprising such a vector, and recombinanttechniques for the production of the antibody.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Standardtechniques for cloning and transformation may be used in the preparationof cell lines expressing the antibodies of the present invention.

Vectors

Many vectors are available. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Recombinantexpression vectors containing a nucleotide sequence encoding theantibodies of the present invention can be prepared using well knowntechniques. Expression vectors may include a nucleotide sequenceoperably linked to suitable transcriptional or translational regulatorynucleotide sequences such as those derived from mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includetranscriptional promoters, operators, enhancers, mRNA ribosomal bindingsites, and/or other appropriate sequences which control transcriptionand translation initiation and termination. Nucleotide sequences are“operably linked” when the regulatory sequence functionally relates tothe nucleotide sequence for the appropriate polypeptide. Thus, apromoter nucleotide sequence is operably linked to, e.g., the antibodyheavy chain sequence if the promoter nucleotide sequence controls thetranscription of the appropriate nucleotide sequence. An example of auseful expression vector for expressing the antibodies of the presentinvention may be found in PCT Publication No. WO 04/070011, which isincorporated herein by reference.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody. The signal peptide may be cleaved from thepolypeptide upon secretion of antibody from the cell. Examples of suchsecretory signals are well known and include, e.g., those described inU.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.

Host Cells

Host cells useful in the present invention are prokaryotic, yeast, orhigher eukaryotic cells and include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., Baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

Prokaryotes useful as host cells in the present invention include gramnegative or gram positive organisms such as E. coli, B. subtilis,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, andShigella, as well as Bacilli, Pseudomonas, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446), althoughother strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E.coli W3110 (ATCC 27,325) are suitable. These examples are illustrativerather than limiting.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec,Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA) and pRSET(Invitrogen Corporation, Carlsbad, Calif., USA) series of vectors(Studier, J Mol Biol 219:37 (1991); Schoepfer, Gene 124:83 (1993)).Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include T7, (Rosenberg, et al., Gene 56:125 (1987));β-lactamase (penicillinase), lactose promoter system (Chang, et al.,Nature 275:615 (1978), Goeddel, et al., Nature 281:544 (1979));tryptophan (trp) promoter system (Goeddel, et al., Nucl Acids Res 8:4057(1980)); and tac promoter (Sambrook, et al., Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990)).

Yeasts or filamentous fungi useful in the present invention includethose from the genus Saccharomyces, Pichia, Actinomycetes,Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora,and filamentous fungi such as Neurospora, Penicillium, Tolypocladium,and Aspergillus. Yeast vectors will often contain an origin ofreplication sequence from a 2p yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman, et al., J Biol Chem 255:2073 (1980)) or otherglycolytic enzymes (Holland, et al., Biochem 17:4900 (1978)) such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer, etal., Gene 107:285 (1991). Other suitable promoters and vectors for yeastand yeast transformation protocols are well known in the art. Yeasttransformation protocols are well known. One such protocol is describedby Hinnen, et al., Proc Natl Acad Sci 75:1929 (1978). The Hinnenprotocol selects for Trp⁺ transformants in a selective medium.

Mammalian or insect host cell culture systems may also be employed toexpress recombinant antibodies. In principle, any higher eukaryotic cellculture is workable, whether from vertebrate or invertebrate culture.Examples of invertebrate cells include plant and insect cells (Luckow,et al., Bio/Technology 6:47 (1988); Miller, et al., GeneticsEngineering, Setlow, et al., eds. Vol. 8, pp. 277-9, Plenam Publishing(1986); Mseda, et al., Nature 315:592 (1985)). For example, Baculovirussystems may be used for production of heterologous proteins. In aninsect system, Autographa californica nuclear polyhedrosis virus (AcNPV)may be used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter). Other hosts that have been identifiedinclude Aedes, Drosophila melanogaster, and Bombyx mori. A variety ofviral strains for transfection are publicly available, e.g., the L-1variant of AcNPV and the Bm-5 strain of Bombyx mori NPV, and suchviruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Moreover, plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can also be utilized as hosts.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) has become a routine procedure. See Tissue Culture,Kruse, et al., eds., Academic Press (1973). Examples of useful mammalianhost cell lines are monkey kidney; human embryonic kidney line; babyhamster kidney cells; Chinese hamster ovary cells/-DHFR (CHO, Urlaub, etal., Proc Acad Sci USA 77:4216 (1980)); mouse sertoli cells; humancervical carcinoma cells (HELA); canine kidney cells; human lung cells;human liver cells; mouse mammary tumor; and NSO cells.

Host cells are transformed with the above-described vectors for antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, transcriptional and translationalcontrol sequences, selecting transformants, or amplifying the genesencoding the desired sequences. Commonly used promoter sequences andenhancer sequences are derived from polyoma virus, Adenovirus 2, Simianvirus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derivedfrom the SV40 viral genome may be used to provide other genetic elementsfor expression of a structural gene sequence in a mammalian host cell,e.g., SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites. Viral early and late promoters are particularlyuseful because both are easily obtained from a viral genome as afragment which may also contain a viral origin of replication. Exemplaryexpression vectors for use in mammalian host cells are commerciallyavailable.

The host cells used to produce the antibodies of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) aresuitable for culturing host cells. In addition, any of the mediadescribed in Ham, et al., Meth Enzymol 58:44 (1979), Barnes, et al.,Anal Biochem 102:255 (1980), and U.S. Pat. Nos. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as X-chlorides,where X is sodium, calcium, magnesium; and phosphates), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Also included in the present invention are polypeptides comprising aminoacid sequences that are at least about 70% identical, preferably atleast about 80% identical, more preferably at least about 90% identicaland most preferably at least about 95% identical (e.g., 95%, 96%, 97%,98%, 99%, 100%) to the amino acid sequences of the antibodies providedherein when the comparison is performed by a BLAST algorithm wherein theparameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences. Polypeptides comprising amino acidsequences that are at least about 70% similar, preferably at least about80% similar, more preferably at least about 90% similar and mostpreferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%,100%) to any of the reference amino acid sequences when the comparisonis performed with a BLAST algorithm wherein the parameters of thealgorithm are selected to give the largest match between the respectivesequences over the entire length of the respective reference sequences,are also included in the present invention.

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned. Sequence similarity includes identical residuesand non-identical, biochemically related amino acids. Biochemicallyrelated amino acids that share similar properties and may beinterchangeable are discussed above.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., (1990) J.Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet.3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141;Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang,J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993)Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl.Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “Amodel of evolutionary change in proteins.” in Atlas of Protein Sequenceand Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp.345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M.,et al., “Matrices for detecting distant relationships.” in Atlas ofProtein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., etal., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl.Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol.Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

In another embodiment, the invention relates to an isolated nucleic acidor nucleic acids, for example DNA, encoding the polypeptide chains ofthe isolated antibodies or antigen binding fragments of the invention.In one embodiment, the isolated nucleic acid encodes an antibody orantigen binding fragment thereof comprising at least one mature antibodylight chain variable (VL) domain and at least one mature antibody heavychain variable (VH) domain, wherein the VL domain comprises at leastthree CDRs having the sequence of SEQ ID NO: 3, and the VH domaincomprises at least three CDRs having the sequence of SEQ ID NO: 1. Insome embodiments, the isolated nucleic acid encodes both a light chainand a heavy chain on a single nucleic acid molecule, and in otherembodiments, the light and heavy chains are encoded on separate nucleicacid molecules. In another embodiment, the nucleic acids further encodea signal sequence.

Methods Of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative, or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody or a fragment of the antibody.Once a polynucleotide encoding an antibody molecule has been obtained,the vector for the production of the antibody may be produced byrecombinant DNA technology. An expression vector is constructedcontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. In one aspect of theinvention, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention as described above. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. Bacterial cells such as E. coli, and eukaryoticcells are commonly used for the expression of a recombinant antibodymolecule, especially for the expression of whole recombinant antibodymolecule. For example, mammalian cells such as CHO, in conjunction witha vector such as the major intermediate early gene promoter element fromhuman cytomegalovirus, are an effective expression system for antibodies(Foecking, et al., Gene 45:101 (1986); Cockett, et al., Bio/Technology8:2 (1990)).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, COS, 293, 3T3, or myelomacells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for one to two days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, etal., Proc Nail Acad Sci USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes,which can be employed in tk, hgprt or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare, et al., Proc NatlAcad Sci USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan, et al., Proc Natl Acad Sci USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu,et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance tohygromycin (Santerre, et al., Gene 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel, et al., eds., Current Protocols in MolecularBiology, John Wiley & Sons (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press (1990); and in Chapters12 and 13, Dracopoli, et al., eds, Current Protocols in Human Genetics,John Wiley & Sons (1994); Colberre-Garapin, et al., J Mol Biol 150:1(1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington, et al., “The use of vectorsbased on gene amplification for the expression of cloned genes inmammalian cells,” DNA Cloning, Vol. 3. Academic Press (1987)). When amarker in the vector system expressing antibody is amplifiable, increasein the level of inhibitor present in the culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse, et al., Mol Cell Biol 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, ProcNatl Acad Sci USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and size-exclusion chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification. See, e.g., PCTPublication No. WO 93/21232; European App. No. EP 439,095; Naramura, etal., Immunol Lett 39:91 (1994); U.S. Pat. No. 5,474,981; Gillies, etal., Proc Nail Acad Sci USA 89:1428 (1992); Fell, et al., J Immunol146:2446 (1991), which are incorporated by reference in theirentireties.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide (SEQ ID NO: 18), such as the tag provided ina pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311), among others, many of which are commercially available. Asdescribed in Gentz, et al., Proc Natl Acad Sci USA 86:821 (1989), forinstance, hexa-histidine (SEQ ID NO: 18) provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson, et al., Cell 37:767 (1984)) and the “flag” tag.

Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, may beremoved, for example, by centrifugation or ultrafiltration. Carter, etal., Bio/Technology 10:163 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody is secreted into the medium, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or MilliporePellicon ultrafiltration unit. A protease inhibitor such as PMSF may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human IgG1, IgG2 or IgG4heavy chains (Lindmark, et al., J Immunol Meth 62:1 (1983)). Protein Gis recommended for all mouse isotypes and for human IgG3 (Guss, et al.,EMBO J5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered. Following anypreliminary purification step(s), the mixture comprising the antibody ofinterest and contaminants may be subjected to low pH hydrophobicinteraction chromatography using an elution buffer at a pH between about2.5-4.5, preferably performed at low salt concentrations (e.g., fromabout 0-0.25M salt).

Antibodies to mIQM of B-Cell Receptor Complex

The present invention relates to antibodies specifically targeting themembrane bound IgM (mIgM) component of the B-cell Receptor Complex(BCRC). As the majority of B-cell lymphomas and leukemias express mIgMon their cell surface, these antibodies can be used in the study of thismolecule and the diagnosis and treatment of mIgM associated diseases.

The B-cell Receptor Complex is the central signaling element of theB-cell arm of the immune system controlling differentiation, cell growthand apoptosis. This cell surface molecular complex is expressed andconstitutively activated in all B-cell malignancies (Tsubata T, et al.,B cell signaling. Introduction. 20:675-678 (2000); Gauld S B, et al.,Science 296:1641-1642 (2002); Girurajan M, et al., J Immunol15:5715-5719 (2006)). The BCRC consists of a trans-membrane version ofthe secreted form of Ig (the receptor), closely associated with CD79c4(the signaling element) (See, Reth M, Nature 338:383-384 (1989); Gold MR. et al., Proc Natl Acad Sci USA 88:3436-3440 (1991); Jugloff L S. etal., J Immunol 159:1139-1146 (1991); Cambier J C, et al., FASEB J6:3207-3217 (1992); Flaswinkel H, et al., EMBO J 13:83-89 (1994);Burkhardt A L, et al., Mol Cell Biol 14:1095-1103 (1994); Rowley R B, etal., J Biol Chem 270:11590-11594 (1995); Kabak S, et al., BiochemBiophys Res Commun 324:1249-1255 (2004); Patterson H C, et al., Immunity25:55-65 (2006); Polson A G, et al., Blood 110:616-623 (2007)). A smallextracellular peptide segment (extracellular proximal domain, ECPD orPD) is also present between the trans-membrane sequence of the mIg andthe homologous Ig consensus sequence present in both the membrane andsecreted form of the Ig (Bestagno M, et al., Biochemistry 40:10686-10692(2001); Poggianella M, et al., J Immunol 177:3597-3605 (2006)). This PDis unique for each membrane Ig class and it is not present on thecorresponding secreted Ig form. In addition, based on a search of thehuman genome database, the PD sequence for each Ig membrane class isunique with respect to all sequences contained therein and compared tothe reported corresponding murine sequences. Thus, a genome data banksearch of the specific sequence for each of the class-specific PDsyielded only its corresponding membrane Ig class. No other membraneproteins could be identified that use these sequences as determined bythe gene bank searches. In addition, no homologous peptide sequenceswere found to suggest the evolutionary derivation of these smalldomains.

Because of the presence of large amounts of Ig in circulating blood, thehomologue membrane bound Ig (mIg) was not thought to be a relevanttarget for drug development. The present application shows that the BCRCcan be targeted without interference from circulating Ig by targetingthe short peptide linkers that present extracellularly between thetrans-membrane hydrophobic amino acid sequence and the consensussecreted Ig homologue sequence, i.e., PD. In addition, mRNA splicevariants comprising the mIgM constant domain 4 (μC4) provide newimportant specific epitopes. These findings led to the generation hereinof anti-PD mAbs, i.e., anti-mIgM and anti-mIgG PD mAbs and an antibodybinding a unique μC4 epitope. These mAbs target mIgM or mIgGclass-specific BCRCs and thereby can be used to purify these Igreceptors, and to further explore and discover other neo-antigenspresent in the mIg compared to the corresponding serum version. Becauseof their unique sequences, it is hypothesized that anti-PD mAbs couldmodulate downstream signaling pathways in the signal-transduction frommIg to CD79αβ if the signal is mediated through PDs. An advantage ofthis targeting approach of utilizing anti-PD mAbs is that disruption ofdownstream pathways associated with BCRC would be expected to bemodulated only in B-cells and restricted to the targeted Ig class, e.g.,mIgM expressing cells only. In addition to the specific epitopescontained in the PD, the adjacent or proximal sequence contained withinthe constant region 4 of IgM, μC4, is truncated in the mIgM compared tothe sIgM by deletion of the 20 proximal amino-acids of this domain.Thus, one would expect to detect additional immunologically definedneo-epitopes further distinguishing mIgM from sIgM in mIgM constantregion 4, μC4. The truncation is also responsible for loss of aglycosylation site; the J-Chain binding is absent and this region isproximal to the mIg clustering site localized in the μC4 domain (See,Tolar P et al, Immunity 30(1):44-55 (2009)). It became clear that mAbscould be generated to neo-epitopes in mIgM constant domain 4 and onesuch mAb was isolated that also modulates signaling through the BCRC. AsmIgM is the receptor component of the BCRC, it must transmit theactivation signal to CD79αβ where the intra-cellular phospho-kinasesreside. The exact point of signal transmission from one molecule to theother is still not known.

With a goal to specifically modulate the BCRC, specific monoclonalantibodies (mAbs) targeting the mIg molecule at its PD, anti-PD mAbs,were generated. A large panel of peptide-specific mAbs detecting the13-mer peptide PD sequence (EGEVSADEEGFEN) (SEQ ID NO: 11) specific forthe mIgM molecule and the 18-mer peptide PD sequence(ELQLEESCAEAQDGELDG) (SEQ ID NO: 12) specific for mIgG were generatedand found to have peptide specific binding and cell binding for peptideexpressing cells. High affinity anti-PD monoclonal antibodies (mAbs)were generated by immunization techniques described below. Thesemonoclonal antibodies were shown by ELISA, Western blots and ScanningImmuno-Electron Microscopy (SEM) to bind to mIgM protein and mIgM+expressing cell lines CA 46 (CRL 1648), SU-DHL-5 (CRL 2958), Ramos(CRL-1596), Namalwa (CRL-1432), ST 486 (CRL-1647), MC 116 (CRL-1649),and HT (CRL-2260). Using these high affinity anti-PD monoclonalantibodies, mIgM was immune-affinity purified and used to immunize mice.Second generation antibodies detecting conformational epitopes on BCRCand not reacting with slgM in ELISA/Western/SEM assays were collected.Growth inhibition was assessed by MTT/CASPASE and clonogenic limitingdilution assays as described below. Four monoclonal antibodiesdesignated mAb1-1, mAb2-2b, mAb3-2b and mAb4-2b were selected forfurther studies.

In the course of generating and assessing these specific mAbs to mIgMPD/purified mIgM, several issues and challenges arose:

1. Initial clones collected were of low affinity despite the uniquesequence of the antigen target. Screening immunized Balb/c micedemonstrated that the sera responses were poor. Various adjuvants wereinvestigated without achieving measurable increased titer or affinity ofthe sera samples. Various strains of mice were subsequently tested andonly CD-1 mice were found to be appropriate hosts for generating highaffinity antibodies.

2. The target polypeptide exists in at least three isomeric forms. Togenerate clinically appropriate mAb reagents, mAbs were screened forthose mAbs that recognized all isomeric forms of the target PD.

3. In an effort to enhance the affinity of the murine antibody immuneresponse, and to broaden the search for additional epitopes, purifiedmIgM preparations from cell extracts (acquired from a mAb1-1immuno-affinity column) were incorporated into the immunogen (byco-administration with mIgM PD-peptide-MAP immunogen).

4. Antibody mAb1 was derived from a fusion comprising a mIgM-PDpeptide-MAP immunogen only, whereas all other antibodies (mAb 2, mAb 3and mAb 4) were generated using the purified mIgM in addition to a mIgMPD-peptide-MAP immunogen. To indicate that these mAbs were derived fromfusions using different immunogens, the suffix 1 was added to mAb1(mAb1-1) and the suffix 2 was added to the other antibodies (mAb2-2,mAb3-2 and mAb2-4). Additionally, since the purified mIgM used togenerate the antibodies mAb2, mAb3 and mAb4 was derived from cell line b(CA 46, CRL 1648) extract, the letter b was added to the suffix 2(mAb2-2b, mAb3-2b and mAb4-2b).

5. During the immunizations with purified mIgM, mAbs were generated toan epitope shared with the 4th constant region of mIgM that was notdetected on serum IgM. This is a consequence of neo-antigens generateddue to the terminal deletion of the distal 20 amino acids in themembrane IgM as compared to serum IgM mRNA splice variants. mAb4-2bproves that structure changes in the 4th constant region of mIgM inducedunique conformation epitopes in the IgM domain μC4.

The following studies, which were required to prove mAb specificity andto investigate critical aspects of the biologic activity, are presentedlater in the application in the following order:

1. Generation of and selection of hybridoma panels

2. Specificity studies on live target cells and cell extracts (Table 4)

3. Specificity studies for target peptide, isomers and cell extracts(Tables 5, 6)

4. Molecular specificity studies of inhibition of direct binding toimmune-affinity mIgM or Perfect-FOCUS™ extract (Tables 7, 8).

5. Molecular epitope mapping by competitive mAb binding and 6-merinhibition (Tables 9, 10)

6. Scanning Electron Microscopy binding studies (Tables 11-18, FIGS.1-5)

7. mAb binding mediated BCRC internalization (Tables 19, FIG. 2)

8. mAb4-2b mediated growth inhibition, anti-clonogenic activity andapoptosis (Tables 20-24 and FIGS. 6-7)

As shown later in the present application, cell surface binding assaysdemonstrated the specificity of these monoclonal antibodies by testingagainst a panel of mIgM+vs mIgM−(mIgG+) live cells/fixed cells orextracts. In addition, normal or Waldenstrom's Macroglobulinemia serafailed to block or reduce mAb binding to mIgM+ cells, results which werealso confirmed by SEM. These two studies clearly demonstrate achievementof the ability to target B-cells specifically, in vivo.

At 37° C., anti-PD monoclonal antibodies (mAb1-1, mAb2-2b, mAb3-2b)internalized mIgM (BCRC) by 30 mins, but they did not modulate cellgrowth inhibition. Second generation mAb4-2b also mediated mIgM (BCRC)internalization, but additionally, in low density cultures, cell growthinhibition, anti-clonogenic activity and apoptosis were observed.Apoptosis was seen in a variety of malignant B-cell lines including highand low mIgM/CD79αβ expressing cells.

No antibodies have been reported that (1) bind mIgM expressing cellsspecifically and (2) do not react with human serum IgM, and have highenough avidity to immune-affinity purify mIgM. Commercial preparationsof mIgM are of low quality. The present invention provides suchantibodies capable of mIgM purification despite the presence of serum orsecreted IgM in the cytoplasm of these B-cells.

The data presented below demonstrate that BCRC internalization wasinsufficient to interrupt the BCRC signaling cascade as evidenced bycell growth inhibition assays. Despite the lack of detectable residualmIgM and CD79αβ on the cell surface, no growth inhibition was observed.These data strongly suggest that signal transduction from mIgM to CD79αβis not mediated through the PD peptide sequence and internalized BCRCcontinues to display phosphorylated CD79αβ in its internalizedcompartment. Upon mAb4-2b binding to a non-ligand binding site on mIgM,mAb4-2b induced both BCRC internalization and in another distinct event,growth inhibition and apoptosis. By competitive mAb studies, theapoptosis mediating conformational epitope appears to be shared, butresides predominantly outside of the linear PD sequence. As mAb4-2bshows increased binding to purified mIgM compared to the linear mIgM-PDpeptide and its binding is not substantially blocked by soluble PD6-mers, the mAb4-2b target epitope is either conformational, or constantregion 4 influences its binding, or its epitope resides predominantly inconstant region 4. The experimental results support the use of thesemonoclonal antibodies for drug/radioisotope targeting vehicles or as amediator of inhibition of the BCRC signaling pathway. As these agentshave a high level of specificity because they do not bind to non-mIgMB-cells, normal lymphocytes and non-lymphatic tissues may be sparedtoxicity.

Example 1 Generation of Anti-ECPD Hybridoma Panels

To isolate monoclonal antibodies reactive with the target peptides,IgM-EGEVSADEEGFEN (SEQ ID NO: 11) and IgG-ELQLEESCAEAQDGELDG (SEQ ID NO:12), immunogens carrying these peptides were generated(glutathione-S-transferase (GST)) or purchased (Multiple Antigen Peptide(MAP) (Bio-Synthesis, Lewisville, Tex.) and KLH-peptide (Bio-Synthesis,Lewisville, Tex.)). GST, MAP and KLH constructs were used as immunogeniccarrier proteins and sets of mice were immunized with one orcombinations of the proteins carrying the target peptide. With initialexperiments utilizing Balb/c mice as hosts, it became immediately clearthat these peptides were not immunogenic, even with standard commercialadjuvant preparations. Low immunogenicity is consistent with previousefforts to produce anti-mIgM and mIgG PD mAbs, while in contrast,efforts to produce mIgE have resulted in several functionally distinctversions (Poggianella M, et al., J Immunol 177:3597-3605 (2006);Feichter S, et al., J of Immunol 180:5499-5505 (2008)). The first panelof 11 monoclonal antibodies generated from Balb/c mice were deemed tooweakly reactive to potentially be of clinical value.

It was discovered that of all the mouse strains tested (Balb/cauto-immune mice strains, etc.), only CD-1 mice were capable ofsignificant immune response to these immunogens. Control free KLH(Sigma-Aldrich, St. Louis, Mo., USA) was also obtained for in vitroassays. In addition, the mIgM and mIgG fractions were collected byPerfect-FOCUS™—membrane protein extraction technology (G Biosciences, StLouis, Mo., USA), which yielded enriched mIgM or mIgG preparations fromhuman cell lines, MC 116 (CRL 1649) (Undifferentiated lymphomaexpressing mIgM), CA 46 (CRL 1648) (Burkitt's lymphoma expressing mIgM),ST 486 (CRL 1647) (Burkitt's/CLL like cell line expressing mIgM), HT(CRL 2260) (Diffuse mixed B-cell lymphoma expressing mIgM) or DB (CRL2289) (Large B-cell lymphoma expressing mIgG) adequate for initial ELISAstudies. These enriched fractions of membrane IgM or IgG were used toimmunize sets of CD1 mice, which generated the second generationmonoclonal antibody mAb4-2b. Using various adjuvants, pre- andpost-immunization serum were collected and diluted 1:100 and tested forpeptide specific activity with ELISA.

New immunization strategies were developed. Extending the peptides intothe μC4 domain 4 region (“extended peptide” 18 mer), to possibly captureconformational epitopes using highly purified target protein for boostsand using CD1 mice and new adjuvants, resulted in six mice with postimmunization serum titers of >1:10,000. These mice were selected forhybridoma generation using standard techniques. Four clones wereisolated that were active in binding to peptide/extended peptide andmembrane extraction fractions of mIgM in ELISA, and two clones werespecific for mIgG. The mIgM/mIgG molecules were proven to be expressedby CRL 1647 or CRL 2289, respectively, using primers specific for thetwo PDs by RT PCR and shown to be present in the cell membrane fractionby Western blot analysis. For further screening purposes, constructs(GST, MAP and KLP+/− peptide or extended peptide) were collected for PDpeptide mIgM or mIgG, and also for control peptides such as the commonoverlapping sequences of mIgE and peptide mIgA. mIgD peptide was used inthis part of the analysis as all B-cell extracts contain this peptide aswell. Only immunizations with certain combinations of immunogens andboosted with the membrane extract fraction, yielded clones with goodreactivity; one IgG2b clone produced by a hybridoma cell line fromfusion 117 (mAb1-1) PD-KLH peptide immunization only, two IgG2b clonesproduced by a hybridoma cell line from fusion 118 (mAb2-2b, mAb3-2b),and one IgG1 clone produced by a hybridoma cell line from fusion 119(mAb4-2b).

The initial screen of the hybridoma supernatants required reactivitywith KLH carrying the appropriate PD peptide mIgM or mIgG, no reactivitywith free KLH, normal human serum, purified preparation of IgM and KLHcarrying peptides for IgE, IgD and IgA. To test cell extracts of CRL1647-mIgM and CRL 2289-mIgG, a specific “murine Ig-adsorbed” goatanti-human IgMFc or goat anti-IgGFc anti-sera capture antibody wasattached to solid phase plastic. The NP-40 lysate of CRL 1647 or CRL2289 or control human serum or control breast cancer cell lysate BT-474was bound to the wells. The CRL 1647 lysate provided human mIgM and CRL2289 provided human mIgG to bind the capture antibody and the wells werethen washed three times. Hybridoma supernatants were then added where“specific” monoclonal antibodies bound to the captured human mIgM ormIgG, which was bound by the plastic bound captured anti-human IgMFc orIgGFc, to form a goat anti-human-IgMFc-mIgM-mAb complex or goatanti-human-IgGFc-mIgG-mAb complex. The mouse mAb was then detected withspecific goat anti-mouse Ig HRP labeled (preabsorbed with human IgM orhuman IgG). Other positive cell extracts, such as Namalwa (CRL 1432) andCA 46 (CRL 1648), yielded similar results.

Monoclonal antibodies collected into a panel included monoclonalantibodies designated mAb1-1, mAb2-2b, mAb3-2b, mAb4-2b, and mAb11-1.Monoclonal antibody mAb1-1 is an IgG2b isotype. Monoclonal antibodymAb1-1 was produced by a hybridoma cell line from fusion 117. Monoclonalantibodies mAb2-2b and mAb3-2b are IgG2b isotypes and mAb4-2b is an IgG1isotype. Class switch variants were developed to attain IgG1 isotypesfor certain experiments to diminish non-specific cell Fc receptorbinding present both on target cells and in cell extracts. Monoclonalantibodies mAb 2-2b and mAb3-2b were produced by a hybridoma cell linefrom fusion 118, and mAb4-2b was produced by hybridoma cell line fromfusion 119. Monoclonal antibody mAb11-1 is an IgG1 isotype. Monoclonalantibody mAb11-1 was produced by a hybridoma cell line from fusion 200and is reactive with mIgG. A second anti-mIgG mAb was also collected butnot used in the experiments described herein.

Specificity of reactivity was further confirmed using human mIgE derivedfrom human B-cell line SK007 (human B-cell line expressing mIgE withoutmIgM) by NP-40 lysis of SK007 cells and tested with ELISA using specificgoat anti-human IgE capture antibody. Taken together, these assaysindicated that the monoclonal antibodies panel recognized in ELISA boththe synthetic peptide sequence contained in the immunogen and thepeptide sequence in the human mIgM or mIgG native protein derived byNP-40 lysis and Perfect-FOCUS™ fraction. To check that they did notreact with transmembrane or cytoplasmic domains, fluorescent microscopy(FM) of viable cells using fluorescent labeled goat anti-mouse Igpre-absorbed with mIgM expressing CLL (chronic lymphocytic leukemia)cells, was used to detect mAb bound to viable CLL cells. Resultsdemonstrated mAb specific, but weak cell membrane, staining as expectedand similar to anti-light chain activity, indicating low level mIgM cellsurface expression. In general, CLL cells express low quantities of BCRCcompared to most B-cell lymphomas. Typically, in the clinic, CLL cellsare determined to be monoclonal populations and hence malignant, bytyping the light chain of mIgM as kappa or lambda. However, it appearedthat all four clones did bind to intact viable cells appropriately inthe Hemadsorption assays as they were strongly positive with all mIgM+cell lines tested.

In addition, CLL cells cultured in the presence of these monoclonalantibodies demonstrated increased complexity by side scatter plots inFlow Cytometry compared to controls or polyclonal anti-IgM, suggesting apossible novel cellular effect.

As a consequence of this initial screening strategy, the clones in thepanel had the following characteristics: positive in ELISA for: (1)KLH-peptide/extended peptide (mIgM) and (2) reactive with NP-40 lysatesof positive expressing cell lines CA46 (CRL 1648) and Namalwa (CRL1432)using capture anti-human IgMFc. The clones were all negative in ELISAwith (1) KLH alone, (2) KLH with irrelevant peptide, (3) breast cancercell line BT-474 NP-40 lysate using capture anti-human IgMFc, (4) humanserum using capture anti-human IgMFc, (5) human Waldenstrom'sMacroglobulinemic serum using capture anti-human IgMFc, and (6) lysateof SP2/0 mouse myeloma cell line and H2.8 (CRL 2568) mouse myeloma cellline as controls (HRP-labeled mAb). However, testing by ELISA reactivityto the membrane extract of CA 46 (Perfect-FOCUS™, G-Biosciences, St.Louis, Mo.) and Western blot analysis of CA 46 NP-40 lysate demonstrateddifferences among the clones in their capability to recognize or bind totheir epitopes after extraction by Triton™ X-100 and/or exposure to SDS.This suggested that even for the small peptide target 13 mer or theextended 18 mer, peptide conformational changes were critical for mAbbinding. For example, the fact that there were clones that detected themIgM band on Western blots, positive against KLH-peptide orPerfect-FOCUS™ in ELISA, and were reactive in the Triton™ X-100 ELISAsuggests that a subset of these clones recognized an epitope preservedin detergent. In addition, the Western blots were complicated, showingbinding to multiple molecular weight bands related to complexes of mIgMand CD79αβ.

As mIgM is expressed at low levels, the Flow Cytometry would not be areliable methodology to prove negativity, thus scanning electronmicroscopy was done. As radiolabeling reduced affinity of monoclonalantibodies in some cases (Cesano A, Gayko U, Sem Oncol 30:253-257(2003)), binding and epitope mapping was accomplished with directlylabeled antibodies (HRP, Sigma-Aldrich, St. Louis, Mo., USA) andinhibition with small 6-mer peptides or the extended mIgM PD peptide.

Hybridoma cell lines producing the monoclonal antibodies mAb1-1,mAb2-2b, mAb3-2b and mAb4-2b were deposited on Nov. 12, 2014 with theAmerican Type Culture Collection Patent Depository (10801 UniversityBlvd., Manassas, Va.). The deposits were made under the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and the Regulationsthereunder (Budapest Treaty). The hybridoma cell line producingmonoclonal antibody designated mAb1-1 has been given ATCC deposit number______. The hybridoma cell line producing monoclonal antibody designatedmAb2-2b has been given ATCC deposit number ______. The hybridoma cellline producing monoclonal antibody designated mAb3-2b has been givenATCC deposit number ______. The hybridoma cell line producing monoclonalantibody designated mAb4-2b has been given ATCC deposit number ______.

Example 2 Antibody Purification

Antibody purification was accomplished using protein A sepharose columns(Pierce Inc., Rockford, Ill., USA) with application of supernatants fromeach hybridoma line at pH 8.6 in Tris solution. Further washing was donein PBS at pH 7.0 and then eluting mIgM at pH 4.0. Sterile filteredmonoclonal antibodies were stored at 4° C. at 500 micrograms/ml insterile PBS with/without azide. Antibody preparations for ADCC,Complement lysis, growth inhibition assay and biologic assays weresterile filtered in PBS without azide at 1000 micrograms/ml.

Example 3 Antibody Immune-affinity Purification of mIgM

Antibody purification of mIgM was accomplished using mAb1-1 or mAb1-1with mAb2-2b and mAb3-2b covalently bound immune-affinity beads (PierceInc., Rockford, Ill., USA), application of CRL-1648 NP-40 extract andthen washing beads at pH 8.6 in Tris solution. Further washing was donein PBS at pH 7.0 and then eluting mIgM at pH 4.0. Sterile filtered mIgMbatches were stored at 4° C. at 500 micrograms/ml in sterile PBSwith/without azide.

Example 4 Specificity Analysis

Hemadsorption tests of the panel of antibodies against an epithelialcell-line bank was done to eliminate clones with non-specific crossreactivity (Rettig W J, et al., Int J Cancer 58:385-392 (1994); KitamuraK, et al., Proc Natl Acad Sci USA 91:12957-12961 (1994); Garin-Chesa P,et al., Int J Oncol 9:465-471 (1996); Rader C, et al., J Biol Chem 275;13668-13676 (2000)). A subset of these epithelial cell lines had beentested by RT-PCR for PD sequence to define true negatives, however, mIgMhas never been reported to be expressed by malignant cells other thanthose of B-cell lineage and the studies herein did not reveal thesesequences in any of the cell lines in the panel. This observation wasextended by RT-PCR of a panel of melanomas and sarcomas for the PD13-mer mIgM and 18-mer mIgG and found no signal demonstrating expressionof this peptide in these non-B-cell lineage cells. Thus, any bindingwould represent cross reactivity and not BCRC antigen detection. Thebinding of antibodies to cell surfaces of tumor cells was detectedmicroscopically by adsorption of erythrocytes coated with anti-mouse Igantibody or protein A. The titer was defined as the highest dilution ofreagent giving maximum rosetting (Rettig W J, et al., Int J Cancer58:385-392 (1994); Kitamura K, et al., Proc Natl Acad Sci USA91:12957-12961 (1994); Garin-Chesa P, et al., Int J Oncol 9:465-471(1996); Rader C, et al., J Biol Chem 275:13668-13676 (2000)). Controlpositive antibodies were contained in a panel of antibodies previouslydeveloped by applicants and colleagues, including mAb A33, mAb 3S193,mAb G250 and mAb F19 (Rettig W J, et al., Int J Cancer 58:385-392(1994); Kitamura K, et al., Proc Natl Acad Sci USA 91:12957-12961(1994); Garin-Chesa P, et al., Int J Oncol 9:465-471 (1996); Rader C, etal., J Biol Chem 275:13668-13676 (2000)). Extensive screening ofmonoclonal antibodies mAb1-1, mAb2-2b, mAb3-2b and mAb4-2b in bindingassays against a panel of human epithelial cell lines: breast (9), lung(9), colon (12), renal (6), prostate (3), ovary (3), melanoma (6) andsarcoma (2) were all negative.

CRL 1647 was a true positive that is weakly plastic adherent butrendered more adherent by pre-coating plates with poly L-lysine. Fornon-adherent target cells, rosetting was assessed by microscopicallyexamining cells on glass slides.

Example 5 mAb Specific Reactivity with Viable Cell Lines and NP-40Lysate Assays

Hem-adsorption assays (HA) using protein G coated huRBCs were scoredusing phase contrast microscopy (100×) as negative (neg), +, ++, or +++.The cell line panel consisted of (1) mIgM-lambda, CRL 1432, CRL 1596,CRL 1649, CRL 3006, CRL 2958, (2) mIgM-kappa, CRL 1647, CRL 1648, CRL2260, (3) mIgG-lambda 2289, (4) mIgG, CRL 2632, and (5) SK007.Epithelial cancer and melanoma cell lines were selected for reactivitywith isotype-matched control mAbs. ESA of CL (ELISA sandwich assay ofcell lysates) was performed using a mouse Ig pre-adsorbed capture goatanti-human IgM constant region serum and detected with a human Igpre-adsorbed goat anti-mouse Ig-HRP. Of note is that all lymphoma lineshave cytoplasmic IgM similar to serum IgM. Thus, this assay does notconfirm or assess mAb specificity to distinguish reactivity between mIgMvs. serum or cytoplasmic IgM. Results are demonstrated in Table 4 below.

TABLE 4 mAb mAb Isotype Isotype IgG1 IgG2 mAb 1 mAb 2 mAb 3 mAb 4control control HA Cell targets mIgM-kappa +++ +++ +++ +++ neg negmIgM-lambda +++ +++ +++ +++ neg neg mIgG-kappa neg neg neg neg neg negmIgG-lambda neg neg neg neg neg neg mIgE neg neg neg neg neg neg Colon(12) neg neg neg neg +++ +++ Breast (9) neg neg neg neg ++/+++ neg Lung(9) neg neg neg neg ++/+++ neg Melanoma (2) neg neg neg neg ++/+++ negESA of CL Cell targets mIgM-kappa +++ +++ +++ +++ neg neg mIgM-lambda+++ +++ +++ +++ neg neg mIgG-kappa neg neg neg neg neg neg mIgG-lambdaneg neg neg neg neg neg mIgE neg neg neg neg neg neg Colon (12) neg negneg neg +++ +++ Breast (9) neg neg neg neg +++ neg Lung (9) neg neg negneg +++ neg Melanoma (2) neg neg neg neg +++ neg

Statistical analysis: Student's t-test was used to assess statisticalvalidity of Elisa data points shown. All data points consist of 12 wellsin each of 3 experiments performed, and representative average valuesare shown. Data for mAb 1, mAb 2, mAb 3, and mAb 4 were demonstrated toexceed p<0.5 for all, +++ or ++ compared to their respective controls.Values that are bolded are statistically significant.

Cell lines: Positive cell lines were authenticated by acquisition fromthe ATCC and are shown in the first 5 rows of each assay, which areidentified as follows: mIgM-kappa=CRL 1647, CRL 1648, CRL 2260;mIgM-lambda=CRL 1432, CRL 1596, CRL 1649, CRL 2958, mIgG-kappa=CRL 2632;mIgG-lambda=CRL 2289; mIgE=SK007. Colon, breast, lung and melanoma celllines were obtained from the ATCC and tested serologically to confirmtheir identity. This non-lymphoid cancer cell line panel of colon,breast, lung and melanoma cell lines consisted of the following celllines:

-   -   (1) Colon: T84, SW1222, Colo 205, Lim 1215, HT-29, DLD-1,        SW1116, SW 620, SW 480, LoVo, HCT-15, HCT-116    -   (2) Breast: BT 474, SK BR7, CaMa-1, BT-20, MCF-7, SK Br-3,        MDA-MB 453, MDA-MB 436, MDA-MB 468.    -   (3) Lung: H64, SW1271, DMS 78, SK-LU-9, NCI H596, A549, NCI        H1105, NCI H69, DMS 53    -   (4) Melanoma: SK MEL-29, MeWo

Example 6 Relative Binding Studies

Using the ELISA assay as described above, purified monoclonal antibodiesmAb1-1, mAb2-2b, mAb3-2b, mAb4-2b, and mAb11-1 were serially diluted1:4, then were added to bind to the human mIgM bound by the captureanti-human IgM. The bound mouse mAb was then detected with specific goatanti-mouse Ig-HRP labeled reagent. Monoclonal antibodies of the sameisotype could be compared to each other. Similar assays were carried outwith other positive targets identified in Tables 5-8 below.

ELISA Assays

TABLE 5 mAb Molecular Specificity and Relative Reactivity by DirectBinding ELISA reading KLH- KLH- CA SU-DHL- CRL- CRL- CRL- CRL- PDm KLHPDg 46 5 1596 1432 1647 1642 mAb1-1 >4.0 0.3 0.3 3.7 >4.0 3.2 3.5 2.93.0 mAb2-2b 3.7 0.2 0.4 3.4 3.4 3.4 3.2 3.1 3.3 mAb3-2b 3.3 0.3 0.3 3.12.7 3.1 3.2 2.8 2.8 mAb4-2b 0.9 0.3 0.2 >4.0 3.8 >4.0 >4.0 3.9 3.7mAb11-1 0.3 0.3 3.2 0.3 0.2 0.3 >4.0 0.2 0.2 Anti- 0.2 0.2 0.23.8 >4.0 >4.0 >4.0 >4.0 >4.0 huIgM

TABLE 6  mAb Molecular Specificity and RelativeReactivity by Direct Binding mAb mAb mAb 1 mAb 2 mAb 3 mAb 4 controlcontrol mIgM-PD- >4.0 >4.0 >4.0 0.5 0.1 0.1 KLH KLH 0.1 0.1 0.1 0.1 0.10.1 mIgM-PD- >4.0 >4.0 >4.0 0.9 0.1 0.1 MAP MAP 0.2 0.2 0.2 0.1 0.1 0.1mIgM-PD >4.0 >4.0 >4.0 0.8 0.1 0.1 mIgG-PD 0.1 0.2 0.2 0.1 0.1 0.1P-Focus 3.1 2.8 2.6 >4.0 0.3 0.2 P-Focus + 3.3 2.5 2.3 >4.0 0.1 0.1 IAmIgM-PD- 3.8 3.2 3.0 0.9 0.1 0.1 Isomer 1 MIgM-PD- 3.2 3.0 3.2 0.7 0.10.1 Isomer 2 mIgM-PD = EGEVSADEEGFEN (SEQ ID. NO: 11) Isomer 1 =EGENSADEEGFEN (SEQ ID NO: 14) isomer 2 = EGEVSEDEEGFEN (SEQ ID NO: 15)

Statistical analysis: Student's t-test was used to assess statisticalvalidity of data points shown. All data points consist of 12 wells ineach of 3 experiments performed, and representative average values areshown. Data for mAb 1, mAb 2, mAb 3, and mAb 4 were demonstrated toexceed p<0.5 for rows 1, 3 5, 7, 8, 9 and 10 compared to theirrespective controls. Values that are bolded are statisticallysignificant. Abbreviations: PD, proximal domain; mIgM, membrane boundIgM; KLH, keyhole limpet hemocyanin; MAP, multiple antigen peptide (ofPD); P-Focus™, Perfect FOCUS™ cell extract; IA, immune-affinity column(mAb 1).

TABLE 7 mAb Molecular Specificity and Relative Reactivity by Inhibitionof Direct Binding ELISA Reading Purified Purified Purified Purified mIgMmIgM mIgM mIgM blocked blocked blocked blocked CRL- Serum Purified SerumPurified by by by by 2632 normal IgM W.M. mIgM KLH-PDm KLH-PDg KLHCOLO-205 mAb1-1 0.2 0.2 0.3 0.3 >4.0 0.7 >4.0 3.9 3.7 mAb2-2b 0.3 0.30.4 0.3 3.4 0.3 3.6 3.5 3.5 mAb3-2b 0.3 0.3 0.3 0.3 3.6 0.4 3.7 3.6 3.4mAb4-2b 0.3 0.3 0.3 0.2 >4.0 3.8 >4.0 3.8 3.9 mAb11-1 3.5 0.4 0.2 0.30.4 0.3 0.3 0.4 0.2 Anti-huIgM 0.7 >4.0 >4.0 >4.0 >4.0 >4.0 >4.0 >4.03.9 KLH-PDm = keyhole limpet hemocyanin-Proximal Domain peptide for IgMKLH = keyhole limpet hemocyanin KLH-PDg = keyhole limpethemocyanin-Proximal Domain Peptide for mIgG CA 46, SU-DHL-5, CRL-1592,CRL-1432, CRL-1647, CRL-1642 = mIgM + B-cell lines CRL-2632 = mIgG +B-cell line Serum W.M. = Serum from patient with Waldenstrom'sMacroglobulinemia Purified mIgM = Immune-Affinity purified mIgM from CA46 using mAb1-1 COLO-205 = Human colon cancer cell line mAb11-1 =monoclonal antibody to PD of mIgG Anti-huIgM = mouse polyclonalantibodies to human IgM

TABLE 8 Serum and Cell Inhibition of mAb Binding to purified mIgM cellextract CRL 1647 Blank Normal Normal CLL CLL W-Ms DLBCL INHL BreastColon FCS serum plasma serum cells serum serum serum serum serum mAb 1+++ +++ +++ +++ 0 +++ +++ +++ +++ ++++ mAb 2 +++ +++ +++ +++ 0 +++ ++++++ +++ +++ mAb 3 +++ +++ +++ +++ 0 +++ +++ +++ +++ +++ mAb 4 +++ ++++++ +++ 0 +++ +++ +++ +++ +++ Anti- +++ + + ++ ++ 0 + + + + hu- IgM +++= no inhibition detected 0 = complete inhibition

Inhibition of mAb binding to Perfect FOCUS™ target cell extract byserum, plasma and CLL cells demonstrated lack of specific antigen inblood capable of blocking mAb binding to target antigen while, incontrast, CLL cells adsorbed mAb thereby reducing mAb binding to target.

Statistical analysis: Student's t-test was used to assess statisticalvalidity of data points shown. All data points consist of 12 wells ineach of 3 experiments performed, and representative average values areshown as a score of 0 to +++. Data for mAb 1, mAb 2, mAb 3, and mAb 4demonstrated no significant reduction of binding except for preadsorption by CLL cells. These results were significantly different frompolyclonal anti-human IgM shown in the bottom row, exceeding p<0.5 fornormal serum, normal plasma, Waldenstrom's Macroglobulenemia sera andlymphoma breast or colon cancer serum. Scoring shown, +++=no statisticalreduction of binding, ++=p>0.05 reduction, +=p>0.01 and 0=no detectablebinding over background. Serum tested included Waldenstrom'sMacroglobulenemia serum (W-Ms) that contained 4.2 grams/deciliter IgM,CLL serum that contained 22 mg/deciliter of IgM, Diffuse Large CellB-cell Lymphoma ABC type (DLBCL) Indolent non-Hodgkin's lymphoma (iNHL).

Example 7 Binding Studies and Epitope Mapping By Peptide Inhibition

In initial radiolabeling studies, the IgG2b monoclonal antibodies didnot label well by radioimmunoreactivity assays (Barendswaard E C, etal., Int J Oncol 12:45-53 (1998); Barendswaard E, et al., J Nucl Med42:1251-1256 (2001); Scatchard plots www.curvefit.com/scatchardplots.htm#transforming_data_to_create_a_scatchard). Protein labelingkits for HRP (Sigma-Aldrich, St Louis, Mo., USA; Pierce Chemicals,Rockford, Ill., USA) were used in a solid phase labeling technique.Using excess unlabeled antibody to block labeled antibody bindingdefined the following groups of clones: 1. those blocking the labeledantibody (same epitope), 2. those not blocking the epitope (differentepitope) and 3. partial blocking (a near epitope or weaker binder). Thisprocess was repeated until all the clones were epitope defined.Additional information was obtained through inhibition assays where themAb was blocked by 6-mers and this data was confirmatory. The results ofthe binding studies are presented in Tables 9 and 10 below.

TABLE 9 Molecular Epitope Mapping by Competitive mAb Binding mAb 1 mAb 2mAb 3 mAb 4 Control mAb Blocked by mAb 1 mIgM-PD 11 88 82 72 neg mIgG-PDneg neg neg neg neg KLH-mIgM-PD 6 94 92 74 neg mIgM-PD-KLH 10 92 91 52neg P-Focus + IA 13 90 89 96 neg Blocked by mAb 2 mIgM-PD 80 2 23 69 negmIgG-PD neg neg neg neg neg KLH-mIgM-PD 67 4 24 68 neg mIgM-PD-KLH 78 234 71 neg P-Focus + IA 78 3 31 96 neg Blocked by mAb 3 mIgM-PD 85 35 963 neg mIgG-PD neg neg neg neg neg KLH-mIgM-PD 79 28 4 72 negmIgM-PD-KLH 87 36 9 62 neg P-Focus + IA 82 39 10 85 neg Blocked by mAb 4mIgM-PD 93 87 84 12 neg mIgG-PD neg neg neg neg neg KLH-mIgM-PD 82 88 9514 neg mIgM-PD-KLH 90 94 85 9 neg P-Focus + IA 95 94 86 9 neg

Statistical analysis: Student's t-test was used to assess statisticalvalidity of data shown. All data points consist of 12 wells in each of 3experiments performed, and representative average values are shown.Statistical significance comparisons include for mIgM-PD, KLH-mIgM-PDand P-Focus+IA each in a test of mAb-HRP reactivity pre-blocked byunlabeled mAb (e.g., mAb1-1-HRP was blocked by unlabeled mAb1-1 andsimilar results for each HRP labeled mAb by its partner mAb, bolded).Data points for mAb 1, mAb 2, mAb 3, and mAb 4 were demonstrated toexceed p<0.5 for each self-blocking vs unblocked (data not shown).Further tests demonstrated mAb 1 did not block mAb 2, mAb 3 or mAb 4;mAb 2 did not block mAb 1 and mAb 4; but mAb 3 was partially reducedcompared to mAb 1 and mAb 4. These results were similar for mAb 3analysis showing partial blocking of mAb 2. mAb 4 analysis demonstratedlack of blocking of mAb1, mAb 2 and mAb 4. Conclusions: For mIgM-PD,KLH-mIgM-PD and P-Focus+IA targets, mAb 1 and mAb 4 each detected adistinct epitope while mAb 2 and mAb 3 detected another defined epitopethat is partially shared. mAb 4 showed relative improved binding topurified mIgM (P-Focus+IA) when compared to the other mAbs.

TABLE 10 Molecular Epitope Mapping by Competitive 6-mer peptide BindingBlocked by 6-mer A mAb 1 mAb 2 mAb 3 mAb 4 Control mAb mIgM-PD 28 45 6181 neg mIgG-PD neg neg neg neg neg P-Focus 19 53 54 90 neg mIgM-PD 84 5750 70 neg mIgG-PD neg neg neg neg neg P-Focus 90 48 57 94 neg

6-mer A peptide=EGEVSA (SEQ ID NO: 16) and 6-mer peptide B=EEGFEN (SEQID NO: 17) were used in molar excess X100 compared to mAb-HRP.Statistical analysis demonstrates that mAb 4 detected a distinct epitopenot blocked by either A or B 6-mer. While mAb1 was not blocked by B, mAb1 was strongly blocked by A and mAb 2 and mAb 3 were partially blockedby both A and B. Limiting factors in peptide inhibition assays includedhydrophobicity characteristics of small peptides fragments.

These studies indicate that mAb 4-2b had increased binding to theextended peptide compared to the 11-mer, but neither 6-mer completelyblocked it. These data suggest that its epitope straddles the ECPD andthe terminal mu domain 4 and that its epitope is conformational and thusnot completely inhibited by the small linear peptides. Of note, theantibody, mAb4-2b, was strongest in the hemadsorption assay and FM.

The other 3 mAbs, mAb1-1, mAb2-2b and mAb3-2b split into two epitopes,one proximal and one distal, within the ECPD, despite the fact thatthere was always some degree of partial blocking with the HRP labeledexperiments. While binding to 3 distinct epitopes could be defined,overlap (partial blocking) does exist. Epitope-specific clones with thehighest avidity and restrictive specificity of this final panel of 4mAbs, each detecting an epitope within the extended ECPD as determinedby peptide inhibition were collected into an “epitope panel” of 3monoclonal antibodies.

Example 8 Binding Studies by Scanning Immuno-Electron Microscopy

Cell line CA 46 (CRL 1648) represents a B-cell cell line with relativelylow mIgM expression based on “DIM” light chain reactivity by flowcytometric analysis, similar to chronic lymphocytic leukemia (CLL)cells, and was used in Scanning Immuno-Electron microscopy (SEM) tostudy the binding of the monoclonal antibodies. SEM results arepresented in Tables 11 to 18 below. The micrograph in FIG. 1A shows thecontrol-IgG2b isotype matched control antibody plus secondary goatanti-mouse Ig-gold. The micrographs in FIGS. 1B and 1C show monoclonalantibody m-Ab4-2b, designated mAb4 in the micrograph, binding to twodifferent cells of CA 46 (CRL 1648) at the same magnification as thecontrol antibody in FIG. 1A. The micrograph in FIG. 1D shows monoclonalantibody m-Ab4-2b, designated mAb4 in the micrograph, binding to a thirdCA 46 (CRL 1648) cell at a higher magnification compared to the controlantibody of FIG. 1A. Bright white spots represent immune goldparticles-goat anti-mouse Ig reacting with the mAb4 monoclonal antibodyon the cell surface. No background goat anti-mouse Ig reactivity wasseen with the control antibody in FIG. 1A, indicating lack ofcross-reactivity with human mIgM and lack of non-specific binding by Fcreceptors on B-cells. From these micrographs, it was estimated that mIgMwas present at 5,000-10,000 molecules per cell. These SEM micrographsshow that mAb4-2b binds to the mIgM on the cell surface.

TABLE 11 Target Cell Line: CA 46 (CRL 1648) Scanning Immuno-ElectronGlutaraldehyde fixation Microscopy (SEM) Antibody Tested Cell LineBinding Results mAb1-1 Positive mAb2-2b Positive mAb3-2b PositivemAb4-2b Positive Mouse anti-human IgM Positive Mouse anti-human Kappalight chain Positive mAb anti-human IgM heavy chain Positive mAbanti-human IgG heavy chain Negative

Direct mAb binding studies demonstrated cell surface binding and thepresence of target mIgM was shown by anti-human IgM, anti-human Kappalight chain, anti-human IgM heavy chain reagents.

TABLE 12 Target Cell Line: CA 46 (CRL 1648) Glutaraldehyde fixationPre-incubation of antibody with human serum 1:10 or human Waldenstrom'sMacroglobuinemia serum 1:10 (4.4 gms Scanning Immuno-Electron IgM/dl)Microscopy (SEM) Antibody Tested Cell Line Binding Results mAb1-1Positive mAb2-2b Positive mAb3-2b Positive mAb3-2b Positive Mouseanti-human IgM Negative Mouse anti-human kappa light chain Negative mAbanti-human IgM heavy chain Negative mAb anti-human IgG heavy chainNegative

mAbs were not blocked from cell surface binding by pre-incubation withhuman Waldenstrom's serum containing high levels of serum IgM. Incontrast, anti-human IgM, anti-human Kappa light chain, and anti-humanIgM heavy chain reagents were blocked, resulting in their lack of cellsurface binding.

TABLE 13 Target Cell Line: CA 46 (CRL 1648) Glutaraldehyde fixation; Preincubation of antibody with excess CRL- Scanning Immuno-Electron 1432(adsorption) (mIgM positive cell line) Microscopy (SEM) Antibody TestedCell Line Binding Results mAb1-1 Negative mAb2-2b Negative mAb3-2bNegative mAb4-2b Negative Mouse anti-human IgM Negative mAb anti-humanIgM heavy chain Negative mAb anti human IgG Heavy chain Negative

mAbs were blocked from cell surface binding by pre-incubation with mIgMexpressing CRL 1432, and anti-human IgM, anti-human Kappa light chain,anti-human IgM heavy chain reagents were blocked as they also bind tomIgM on CRL 1432.

TABLE 14 Target Cell Line: CA 46 (CRL 1648) Glutaraldehyde fixation; Preincubation of antibody with excess Scanning Immuno-Electron ProximalDomain peptide for mIgM Microscopy (SEM) Antibody Tested Cell LineBinding Results mAb1-1 Negative mAb2-2b Negative mAb3-2b NegativemAb4-2b Positive Mouse anti-human IgM Positive mAb anti-human IgM heavychain Positive mAb anti-human IgG heavy chain Negative

Excess mIgM PD blocked mAb 1-1, mAb2-2b and mAb3-2b binding to the cellsurface of CRL 1648, whereas mAb4-2b was not blocked and could bedetected binding to the CRL1648 cell surface.

TABLE 15 Target Cell Line: CA 46 (CRL 1648) Pre-incubation of CA 46cells with mAb1-1 at 37° C. for 30 minutes, followed by glutaraldehydefixation, followed by Scanning Immuno-Electron antibody incubation andthen SEM Microscopy (SEM) Antibody Tested Cell Line Binding ResultsmAb1-1 Negative mAb2-2b Negative mAb3-2b Negative mAb4-2b Negative Mouseanti-human IgM heavy chain Negative Mouse anti-human IgG heavy chainNegative

mAb1-1 mediated internalization of mIgM by 30 minutes, resulting in lackof detection of mIgM on the surface of CRL 1648 cells by mAbs oranti-human IgM heavy chain reagent.

TABLE 16 Target Cell Line: CA 46 (CRL 1648) Pre-incubation of CA 46cells with mAb4-2b at 37° C. for 30 minutes followed by ScanningImmuno-Electron glutaraldehyde fixation and then SEM Microscopy (SEM)Antibody Tested Cell Line Binding Results mAb1-1 Negative mAb2-2bNegative mAb3-2b Negative mAb4-2b Negative Mouse anti-human IgM NegativeMouse anti-human IgM heavy chain Negative Mouse anti-human IgG heavychain NegativemAb4-2b mediated internalization of mIgM by 30 minutes, resulting inlack of detection of mIgM on the surface of CRL 1648 cells by mAbs oranti-human IgM heavy chain reagent.

TABLE 17 Target Cell Line: CA 46 (CRL 1648) Scanning Immuno-ElectronFixation of CA 46 cells with glutaraldehyde Microscopy (SEM) followed byincubation with mAb1-1 Using Anti-Mouse followed by mAb IgG1 AntibodyGold Antibody Tested Cell Line Binding Results mAb1-1 Negative mAb2-2bNegative mAb3-2b Negative mAb4-2b Positive Mouse anti-human IgM heavychain Positive Mouse anti-human IgG heavy chain Negative

Anti-mouse IgG1-Gold reagent detected mAb4-2b, which is a mouse IgG1isotype, bound to the surface of CRL 1648 preincubated with the IgG2bisotype mAb1-1 antibody, indicating that mAb1-1 binds to a differentepitope than mAb4-2b and does not block mAb4-2b.

TABLE 18 Target Cell Line: CA 46 (CRL 1648) Scanning Immuno-ElectronFixation of CA 46 cells with glutaraldehyde Microscopy (SEM) Usingfollowed by incubation with mAb2-2b Anti-Mouse IgG1 followed by mAbAntibody Gold Antibody Tested Cell Line Binding Results mAb1-1 NegativemAb2-2b Negative mAb3-2b Negative mAb4-2b Positive Mouse anti-human IgMPositive Mouse anti-human IgM heavy chain Positive Mouse anti-human IgGheavy chain Negative

Anti-mouse IgG1-Gold reagent detected mAb4-2b, which is a mouse IgG1isotype, bound to the surface of CRL 1648 preincubated with the IgG2bisotype mAb2-2b antibody, indicating that mAb2-2b binds to a differentepitope than mAb4-2b and does not block mAb4-2b.

Example 9 mAb Binding Mediates BCRC Internalization

Scanning Immuno-Electron microscopy (SEM) was performed to detectbinding of monoclonal antibody mAb 4 to cells of cell line CRL 1648.FIG. 2A shows monoclonal antibody mAb 4 binding to a glutaraldehydefixed CRL 1648 cell. FIG. 2B shows mAb 4 binding to micro-clusters ofBCRC. When CRL 1648 cells were incubated with mAb 4 at 37° C. for 30minutes, then fixed and stained with goat-anti-mouse Ig, there was alack of detectable monoclonal antibody mAb 4, shown in FIG. 2C, incontrast to the mAb4 binding seen in FIG. 2A. The lack of detectablemAb4 on the membrane was due to BCRC internalization. When the CRL 1648cells were incubated with mAb 4 at 37° C. for 15 minutes, then fixed andstained with goat-anti-mouse Ig, internalization was incomplete andresidual bound monoclonal antibody mAb 4 was seen in FIG. 2D. When CRL1648 cells were incubated with monoclonal antibody mAb 4 at 37° C. for30 minutes, then fixed and stained with goat-anti-hu-IgM, BCRC was notdetectable, which is shown in FIG. 2E.

Example 10 Inhibition of mAb 4 Binding by Pretreated B Cell Lines Withor Without Acid Wash Assessment of mAb Induced Internalization of mIgM

To determine relative cell surface mIgM levels under various conditions,B-cell lines were either exposed to glutaraldehyde-fixed cells (rows 1and 2 in Table 19) (as per SEM protocol below) or viable cells wereused. Cells were exposed to mAb 4, 10 mcgs/ml at 4° C. for 0 minutes(rows 3 and 4 in Table 19), and for 5 minutes (rows 5 and 6 in Table19), 15 minutes (rows 7 and 8 in Table 19), or 30 minutes (rows 9 and 10in Table 19) at 37° C. Cells were then washed with pH 7.0 PBS or pH 4.00.5M acetate buffer 0.15 N NaCl prior to use in inhibition of mAb 4-HRPbinding assays. Row 1 was set as 100% binding for each cell line and row2 demonstrated acid wash ability to remove cell bound mAb 4 and allowmAb 4-HRP adsorption by cells reducing mAb 4-HRP available for bindingassay. Similar results were seen for cells incubated on ice, indicatingthat both glutaraldehyde fixation and cold reduce mAb mediatedinternalization of mIgM. Timed experiments demonstrated that by 30 minat 37° C., cell inhibition is reduced without a difference between PBSand acetate wash (pH 4.0), suggesting that mIgM is predominantlyinternalized. The results are presented in Table 19 below.

TABLE 19 Cell Inhibition of mAb 4- Cell Treatment CRL CRL CRL HRPbinding to target with prior to adsorption 1648 1647 1596Glutaraldehyde-Fixed PBS 100 100 100 Glutaraldehyde-Fixed Acetate 10 2119 Live cells on Ice PBS 92 95 88 Live cells on Ice Acetate 12 18 20Live 37°(5 min) PBS 77 67 66 Live 37° (5 min) Acetate 26 18 27 Live 37°(15 min) PBS 78 81 72 Live 37° (15 min) Acetate 48 44 56 Live 37° (30min) PBS 70 64 60 Live 37° (30 min) Acetate 66 62 61

Example 11 Biologic Activity mAb4-2b Mediates Growth Inhibition,Anti-Clonogenic Activity and Apoptosis

Due to the uniqueness of the sequences in the mIgM PD and evidence thattrans-membrane cell signaling is conveyed to CD79αβ, examining growthcurves (MTT) and clonogenicity of CA 46 (CRL 1648) cells was done todetermine whether there is a modulation of this process (Kikushige Y, etal., Cancer Cell 20(2):246-59 (2011); Martinez-Climent J A, Haematol95(2): 293-302 (2010); Franken N P, et al., Nature Protocols 1:2315-2319(2006)). Initial testing for single clone survival at limiting dilutionin 96 well plates indicated that three of the monoclonal antibodies hadsome activity. The strongest activity was with the monoclonal antibodymAb4-2b that binds in both the PD and Domain 4 and to a conformationalepitope region. Monoclonal antibody mAb4-2b binds to a partiallydetergent sensitive, paraformaldehyde and a reduction resistant epitope.The other 3 mAbs bind to more proximal epitopes in the PD. Whether thesecell growth inhibitory effects are related to blocking epitopes directlytransmitting signaling or are steric-related due to the large size ofthe mAb and/or to micro-clustering is unclear.

Overall, as shown in the inhibition assays below in Tables 20 to 23, themost potent mAb, mAb4-2b, reduced C A 46 clonogenic capacity 100 fold(Kikushige Y, et al., Cancer Cell 20(2):246-59 (2011); Martinez-ClimentJ A, Haematol 95(2): 293-302 (2010); Franken N P, et al., NatureProtocols 1:2315-2319 (2006)).

Inhibition Assays

Cells are plated in 24 well plates and transferred to 96 well plateswith 1:2 serial dilutions as indicated below in Tables 20 to 23. The MTTassay is carried out, where each value is the average of 8 wells perdilution point. ng=no grown, no viable cells

TABLE 20 CA 46 (CRL 1648) # cells/well 5000 2500 1250 0625 0312 01560078 0039 0019 0008 0004 000 mAb4-2b 2.6 1.3 0.9 0.7 ng ng Ng ng ng ngng ng Control 4.0 4.0 4.0 2.7 1.9 1.6 0.9 0.6 04 0.4 ng ngVisual + + + + + + + + confirmation

TABLE 21 SU-DHL-5 (CRL 2958) # cells/well 5000 2500 1250 0625 0312 01560078 0039 0019 0008 0004 000 mAb4-2b 2.0 1.1 0.7 0.4 ng ng ng ng ng ngng ng Control 4.0 4.0 4.0 3.7 2.9 1.9 0.9 0.6 0.4 0.4 ng ngVisual + + + + + + + + confirmation

TABLE 22 Ramos (CRL 1596) # cells/well 5000 2500 1250 0625 0312 01560078 0039 0019 0008 0004 000 mAb4-2b 3.0 2.1 1.7 0.7 0.3 ng ng ng ng ngng ng Control 4.0 4.0 4.0 3.6 2.7 1.1 0.8 0.8 0.5 0.5 0.3 ngVisual + + + + + + + confirmation

TABLE 23 Namalwa (CRL 1432) # cells/well 5000 2500 1250 0625 0312 01560078 0039 0019 0008 0004 000 mAb4-2b 3.2 2.4 0.9 0.2 ng ng ng ng ng ngng ng Control 4.0 4.0 4.0 4.0 2.6 1.0 0.8 0.8 0.5 0.5 0.3 ngVisual + + + + + + + + confirmation

Example 12 Limiting Dilution Assays and Cell Density Experiments

Limiting dilution assays with or without 1 microgram of mAb 4demonstrated significant cell survival and growth characteristics by day10. The results are presented in Table 24 below. Values are presented as% viable cells of mAb 4 treated/% viable cells of control mAb treated.Note the significant difference in cell growth between the doubling ofmedia volume between 48 and 24 cell culture plates. Cells were platedwith 100 microliters in 96 well plates, 250 microliters in 48 wellplates, and 500 microliters in 24 well plates. Marked inhibition ofgrowth was observed up to 500-1000 cells plated. It is believed thatthis represents the effects on a paracrine growth factor produced bystem cell which are killed by mAb 4. These experiments also suggest thatseveral distinct populations of stem cells exist in differentfrequencies capable of rescuing cell growth at different cell densities.

TABLE 24 10 cells/ 10 cells/ 10 cells/ 50 cells/ 50 cells/ 50 cells/Cell Lines 96 well 48 well 24 well 96 well 48 cells 24 well CRL 1648 3221 <1 67 25 <1 CRL 1647 41 18 <1 57 28 <1 CRL 1596 36 23 <1 66 18 <1

Statistical analysis: Student's t-test was used to assess statisticalvalidity of data points shown. All data points consist of 12 wells ineach of 3 experiments performed, and representative average values areshown. The first analysis is represented by each data point comparing %viable cells of mAb 4 treated/% viable cells of control mAb treated.Each of the 18 data points shown reached statistical level of p<0.5. Thesecond analysis demonstrated statistical differences between the 48 wellviable cell counts and the 24 well paired for each cell line incomparison. These also exceeded p<0.5 in each case. In growth inhibitionstudies, MTT viability counts showed that inhibition was inverselyproportional to the number of cells plated. Similar experiments oncontrol mIgM−, mIgG+ expressing cells did not show any biologic effectsand polyclonal rabbit or goat anti-IgM was not anti-clonogenic. Thissuggests that the specificity determining inhibition is located inneo-epitopes near the cell membrane.

Growth curves: mIgM B-cell lines were grown in the presence of 1 μg/mlof mAb4-2b (as shown in Tables 20 to 23). Cells were plated at 20cells/ml. Plates were collected every 2 days, with viable cellsdetermined by MTT assay. Relative MTT OD was plotted for each timepoint. Apoptosis was scored by the absence of viable cells (Day 10) asdetermined by recloning cultures of surviving cells in the absence ofantibody. The results are presented in FIGS. 6A-6F. Both theisotype-matched control antibody and the control anti-PD mAb 2 did notinduce growth inhibition of the CRL 1648 cell line, shown in FIGS. 6A,6B, and 6D-6F. When a control B-cell line expressing mIgG, CRL 2632, wasused, mAb 4 did not bind to mIgG and did not suppress growth of thiscell line, shown in FIG. 6C. FIGS. 6D-6F show that mAb 4 did inducegrowth inhibition of mIgM expressing B-cell lines CRL 1648, CRL 2958,CRL 1596 and CRL 1432.

The inhibition of growth of mIgM-expressing B-cell line CRL 1648 bymonoclonal antibody mAb 4 over ten days was tested at cell dilutions of20 cells/well, 100 cells/well, 250 cells/well, 500 cells/well and 1,000cells/well. As shown in FIG. 7, monoclonal antibody mAb 4 inhibitedgrowth of mIgM expressing B-cell line CRL 1648 for a ten day period, butnot when the concentration of cells plated was >500 cells/well.

Example 13 Complement Lysis and ADCC

The goal was to assess the immune cytotoxic capability of thesemonoclonal antibodies with regard to human complement (C′) (IgG2, IgG3and IgM) and human effector-cell mediated antibody directedcell-mediated cytotoxicity (ADCC) (IgG2 and IgG3) (Paneerselvam M, etal., J Immunol 136:2534-2541 (1986); Welt S, et al., Clin ImmunolImmunopathol 45:214-229 (1987)). As these monoclonal antibodies aremouse monoclonal antibodies, this analysis was in part serving only tohelp determine if C′ or ADCC was positive with these mouse antibodiesand would therefore be an important factor to retain in clinical productdevelopment of humanized antibodies. While mouse IgG1 monoclonalantibodies may not have the capabilities of effector function due totheir Ig sub-class, the focus is to determine if any individual clonehas exceptional activity.

In the final analysis of isotypes collected from the clones of the finalpanel saved for further analysis based on initial binding studies werefour IgG2b and two IgG1 monoclonal antibodies, which includedanti-mIgM-mAb1-1, mAb2-2b, mAb3-2b, and mAb4-2b. None of these werepositive in the assay as they were done at 37° C. and internalizationoccurred rapidly. As these results were a consequence of the rapidinternalization, they are in sharp contrast with other antibodiesbinding to proximal domain epitopes that are reported to mediate theseimmune mechanisms. These results could also be due to low antigenlevels, resistance factors or isotype (Paneerselvam M, et al., J Immunol136:2534-2541 (1986); Welt S, et al., Clin Immunol Immunopathol45:214-229 (1987)). Rituximab and polyclonal rabbit anti-human IgM wereused as positive controls.

Pharmaceutical Formulations

Therapeutic formulations of a polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),i.e., buffering agents, stabilizing agents, preservatives, isotonifiers,non-ionic detergents, antioxidants, and other miscellaneous additives.See Remington's Pharmaceutical Sciences, 16th edition, Osol, Ed. (1980).Such additives must be nontoxic to the recipients at the dosages andconcentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, and iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers may be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants may be present in a range ofabout 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml toabout 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in a microcapsule prepared, for example, by coascervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly/2-hydroxyethyl-methacrylate,poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C. resulting in a loss of biological activity and possiblechanges in immunogenicity.

Rational strategies can be devised for stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

The amount of therapeutic polypeptide, antibody, or fragment thereofwhich will be effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, it isdesirable to determine the dose response curve and the pharmaceuticalcompositions of the invention first in vitro, and then in useful animalmodel systems prior to testing in humans.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof is administered bysubcutaneous injection. Each dose may range from about 0.5 μg to about50 pg per kilogram of body weight, or more preferably, from about 3 pgto about 30 pg per kilogram body weight.

The dosing schedule for subcutaneous administration may vary form once amonth to daily depending on a number of clinical factors, including thetype of disease, severity of disease, and the subject's sensitivity tothe therapeutic agent.

Diagnostic Uses for Anti-B-Cell mIgM Antibodies

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody, such that covalent attachment does not interfere with bindingto B-cell mIgM. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., bybiotinylation, HRP, or any other detectable moiety.

Antibodies of the present invention may be used, for example, but notlimited to, to purify or detect BCRC, including both in vitro and invivo diagnostic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofBCRC in biological samples. See, e.g., Harlow, et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988),which is incorporated by reference herein in its entirety.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic agent. The antibodies can be useddiagnostically, for example, to detect expression of a target ofinterest in specific cells, tissues, or serum; or to monitor thedevelopment or progression of an immunologic response as part of aclinical testing procedure to, e.g., determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance may becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like.

Techniques for conjugating enzymes to antibodies are described inO'Sullivan, et al., “Methods for the Preparation of Enzyme-AntibodyConjugates for Use in Enzyme Immunoassay,” in Methods in Enzymology,Langone, et al., eds. pp. 147-66, Academic Press (1981). See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g.,digloxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten antibody (e.g., anti-digloxin antibody).Thus, indirect conjugation of the label with the antibody can beachieved.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. See Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158. CRC Press (1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of target in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

Antibodies may be attached to solid supports, which are particularlyuseful for immunoassays or purification of the target antigen. Suchsolid supports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.In this process, the antibodies are immobilized on a solid support suchas SEPHADEX™ resin or filter paper, using methods well known in the art.The immobilized antibodies are contacted with a sample containing thetarget to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the target to be purified, which is bound to theimmobilized antibodies. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, that will release the targetfrom the antibodies.

Labeled antibodies, and derivatives and analogs thereof, thatspecifically bind to B-cell mIgM can be used for diagnostic purposes todetect, diagnose, or monitor diseases, disorders, and/or conditionsassociated with the aberrant expression and/or activity of B-cellmalignancies. The invention provides for the detection of aberrantexpression of B-cell mIgM, comprising (a) assaying the expression ofB-cell mIgM in cells or body fluid of an individual using one or moreantibodies of the present invention specific to B-cell mIgM and (b)comparing the level of gene expression with a standard gene expressionlevel, whereby an increase or decrease in the assayed B-cell mIgMexpression level compared to the standard expression level is indicativeof aberrant expression.

Antibodies may be used for detecting the presence and/or levels ofB-cell mIgM in a sample, e.g., a bodily fluid or tissue sample. Thedetecting method may comprise contacting the sample with a B-cell mIgMantibody and determining the amount of antibody that is bound to thesample. For immunohistochemistry, the sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of B-cell mIgM in B cells or bodyfluid of an individual using one or more antibodies of the presentinvention and (b) comparing the level of gene expression with a standardgene expression level, whereby an increase or decrease in the assayedgene expression level compared to the standard expression level isindicative of a particular disorder.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (see, e.g., Jalkanen, et al., J Cell Biol101:976 (1985); Jalkanen, et al., J Cell Biol 105:3087 (1987)). Otherantibody-based methods useful for detecting protein gene expressioninclude immunoassays, such as the enzyme linked immunosorbent assay(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine, (¹³¹I, ¹²⁵I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In, ¹¹¹In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein, rhodamine, and biotin. Radioisotope-bound isotopesmay be localized using immunoscintiography.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of B-cell mIgM in ananimal, preferably a mammal and most preferably a human. In oneembodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds toB-cell mIgM; b) waiting for a time interval following the administrationpermitting the labeled molecule to preferentially concentrate at sitesin the subject where the polypeptide is expressed (and for unboundlabeled molecule to be cleared to background level); c) determiningbackground level; and d) detecting the labeled molecule in the subject,such that detection of labeled molecule above the background levelindicates that the subject has a particular disease or disorderassociated with aberrant expression of B-cell mIgM. Background level canbe determined by various methods including, comparing the amount oflabeled molecule detected to a standard value previously determined fora particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo imaging is describedin Burchiel, et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments.” Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, Burchiel, et al., eds., Masson Publishing (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. Inanother embodiment, the time interval following administration is 5 to20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (U.S. Pat. No. 5,441,050). In another embodiment, themolecule is labeled with a fluorescent compound and is detected in thepatient using a fluorescence responsive scanning instrument. In anotherembodiment, the molecule is labeled with a positron emitting metal andis detected in the patent using positron emission-tomography. In yetanother embodiment, the molecule is labeled with a paramagnetic labeland is detected in a patient using magnetic resonance imaging (MRI). Inanother aspect, the present invention provides a method for diagnosingwhether a patient has a B-cell lymphoma or leukemia by testing for thepresence of B-cell mIgM in certain patient cells or body fluids. In oneembodiment, the method comprises collecting a cell or body fluid samplefrom a subject, analyzing the body fluid for the presence of B-cellmIgM, comparing the amount to a defined or tested level established fornormal cell or bodily fluid and determining if a patient has a B-celllymphoma or leukemia based upon the level of expression of B-cell mIgMin the body fluid. The defined level of B-cell mIgM may be a knownamount based upon literature values or may be determined in advance bymeasuring the amount in normal cell or body fluids. Specifically,determination of B-cell mIgM levels in certain body fluids permitsspecific and early, preferably before disease occurs, detection ofdiseases in the patient. Diseases that can be diagnosed using thepresent method include, but are not limited to, B-cell malignanciesdescribed herein. In the preferred embodiment, the body fluid isperipheral blood or peripheral blood leukocytes.

The antibody of the present invention can be provided in a kit, i.e.,packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides thedetectable chromophore or fluorophore). In addition, other additives maybe included, such as stabilizers, buffers (e.g., a block buffer or lysisbuffer), and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

Therapeutic Uses of Anti-B-Cell mIgM Antibodies

It is contemplated that the antibodies of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed on the mammal.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) can beused as a therapeutic. The present invention is directed toantibody-based therapies which involve administering antibodies of theinvention to an animal, a mammal, or a human, for treating a B-celllymphoma or leukemia. The animal or subject may be an animal in need ofa particular treatment, such as an animal having been diagnosed with aparticular disorder, e.g., one relating to B-cell lymphomas orleukemias. Antibodies directed against B-cell mIgM are useful for B-celllymphomas or leukemias in animals, including but not limited to cows,pigs, horses, chickens, cats, dogs, non-human primates etc., as well ashumans. For example, by administering a therapeutically acceptable doseof an antibody, or antibodies, of the present invention, or a cocktailof antibodies of the present invention, or in combination with otherantibodies of varying sources, disease symptoms may be reduced oreliminated in the treated mammal.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention as described below (including fragments,analogs and derivatives thereof and anti-idiotypic antibodies asdescribed herein). The antibodies of the invention can be used to treat,inhibit, or prevent diseases, disorders, or conditions associated withaberrant expression and/or activity of B-cell mIgM, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of B-cell mIgM includes, but is not limited to, alleviating atleast one of the symptoms associated with those diseases, disorders, orconditions. Antibodies of the present invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

Anti-B-cell mIgM antibodies of the present invention may be usedtherapeutically in a variety of diseases. The present invention providesa method for preventing or treating B-cell malignancy diseases in amammal. The method comprises administering a disease preventing ortreating amount of anti-B-cell mIgM antibody to the mammal. Theanti-B-cell mIgM antibody binds to B-cell mIgM and inhibits cell growthand induces apoptosis.

The amount of the antibody which will be effective in the treatment,inhibition, and prevention of a disease or disorder associated withaberrant expression and/or activity of B-cell mIgM can be determined bystandard clinical techniques. The dosage will depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibodycan be administered in treatment regimens consistent with the disease,e.g., a single or a few doses over one to several days to ameliorate adisease state or periodic doses over an extended time to prevent allergyor asthma. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 150 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. However, other dosageregimens may be useful. The progress of this therapy is easily monitoredby conventional techniques and assays. An exemplary dosing regimen foran anti-LFA-1 or anti-ICAM-1 antibody is disclosed in PCT PublicationNo. WO 94/04188.

The antibodies of the present invention, which may be in the form of acomposition, should be formulated, dosed and administered in a mannerconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the antibody composition to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat a disease or disorder. The antibody need not be,but is optionally formulated with one or more agents currently used toprevent or treat the disorder in question. The effective amount of suchother agents depends on the amount of antibody present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3IL-7, and IFN-γ), for example, which serve to increase the number oractivity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments, such as immunotherapy,chemotherapy, and radioisotopes.

In a preferred aspect, the antibody is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side effects). Various delivery systems are known and can beused to administer an antibody of the present invention, includinginjection, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the compound,receptor-mediated endocytosis (see, e.g., Wu, et al., J Biol Chem262:4429 (1987)), construction of a nucleic acid as part of a retroviralor other vector, etc.

The anti-B-cell mIgM antibody can be administered to the mammal in anyacceptable manner. Methods of introduction include, but are not limitedto, parenteral, subcutaneous, intraperitoneal, intrapulmonary,intranasal, epidural, inhalation, and oral routes, and if desired forimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intradermal, intravenous,intra-arterial, or intraperitoneal administration. The antibodies orcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection: intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably, thedosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition (See, e.g., U.S. Pat. No. 6,514,496).

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment. This may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository, or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.Preferably, when administering an antibody of the invention, care mustbe taken to use materials to which the protein does not absorb.

In another embodiment, the antibody can be delivered in a vesicle, inparticular, a liposome (see Langer, Science 249:1527 (1990); Treat, etal., Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein, et al., eds., pp. 353-365 (1989); Lopez-Berestein,ibid., pp. 317-27; see generally, ibid.).

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);Buchwald, et al., Surgery 88:507 (1980); Saudek, et al., N Engl J Med321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer, et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen, et al., eds., Wiley (1984); Ranger, et al., JMacromol Sci Rev Macromol Chem 23:61 (1983); see also, Levy, et al.,Science 228:190 (1985); During, et al., Ann Neurol 25:351 (1989);Howard, et al., J Neurosurg 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of the antibodyand a physiologically acceptable carrier. In a specific embodiment, theterm “physiologically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such physiological carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable carriers are described in “Remington's Pharmaceutical Sciences”by E. W. Martin. Such compositions will contain an effective amount ofthe antibody, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In addition, the antibodies of the present invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides, carbohydrates, nucleotides, which include microRNA,and DNA synthetic nucleotides, or toxins. See, e.g., PCT PublicationNos. WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; andEuropean App. No. EP 396,387. An antibody or fragment thereof may beconjugated to a therapeutic moiety such as a cytotoxin (e.g., acytostatic or cytocidal agent), a therapeutic agent, or a radioactivemetal ion (e.g., alpha-emitters such as, for example, 213Bi). Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologues thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin,mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), anti-mitotic agents(e.g., vincristine and vinblastine) and highly toxic drugs (e.g.,calicheamicin).

Techniques for conjugating such therapeutic moieties to antibodies arewell known, see, e.g., Arnon, et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld, et al. (eds.), pp. 243-56 Alan R. Liss(1985); Hellstrom, et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery, 2nd ed., Robinson, et al., eds., pp. 623-53, MarcelDekker (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera, et al., eds., pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies ForCancer Detection and Therapy, Baldwin, et al., eds., pp. 303-16.Academic Press (1985); and Thorpe, et al., Immunol Rev 62:119 (1982).Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate. See, e.g., U.S. Pat. No. 4,676,980.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (see,International Publication No. WO 97/33899), AIM II (see, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi, et al., IntImmunol, 6:1567 (1994)), VEGI (see, International Publication No. WO99/23105); a thrombotic agent; an anti-angiogenic agent, e.g.,angiostatin or endostatin; or biological response modifiers such as, forexample, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g., Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by applicants,pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g., Genbank sequences orGeneID entries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. §1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specificembodiments described herein. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

1-46. (canceled)
 47. A monoclonal antibody that specifically binds tomembrane bound IgM of a B-cell Receptor Complex and clones thereof,wherein the monoclonal antibody is selected from the group consisting ofa monoclonal antibody designated mAb4-2b produced by a hybridoma cellline from fusion 119 (ATCC deposit number PTA-121716), a monoclonalantibody designated mAb1-1 produced by a hybridoma cell line from fusion117 (ATCC deposit number PTA-121719), a monoclonal antibody designatedmAb2-2b produced by a hybridoma cell line from fusion 118 (ATCC depositnumber PTA-121717), and a monoclonal antibody designated mAb3-2bproduced by a hybridoma cell line from fusion 118 (ATCC deposit numberPTA-121718).
 48. The monoclonal antibody of claim 47, wherein themonoclonal antibody is the monoclonal antibody designated mAb4-2bproduced by a hybridoma cell line from fusion 119 (ATCC deposit numberPTA-121716) and clones thereof.
 49. The monoclonal antibody of claim 47,wherein the monoclonal antibody is the monoclonal antibody designatedmAb1-1 produced by a hybridoma cell line from fusion 117 (ATCC depositnumber PTA-121719) and clones thereof.
 50. The monoclonal antibody ofclaim 47, wherein the monoclonal antibody is the monoclonal antibodydesignated mAb2-2b produced by a hybridoma cell line from fusion 118(ATCC deposit number PTA-121717) and clones thereof.
 51. The monoclonalantibody of claim 47, wherein the monoclonal antibody is the monoclonalantibody designated mAb3-2b produced by a hybridoma cell line fromfusion 118 (ATCC deposit number PTA-121718) and clones thereof.
 52. Themonoclonal antibody of claim 47, wherein the antibody is a Fab, Fab′,F(ab′)₂, Fd, single-chain Fv, single-chain antibody, disulfide-linkedFv, single domain antibody, antigen binding fragment, diabody, triabody,or minibody.
 53. A monoclonal antibody or antigen binding fragmentthereof that specifically binds membrane bound IgM of a B-cell ReceptorComplex comprising a heavy chain variable region and a light chainvariable region, wherein: (a) the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO: 2 (heavy chain); and (b) the lightchain variable region comprises the amino acid sequence of SEQ ID NO: 4(light chain).
 54. A monoclonal antibody or antigen binding fragmentthereof that specifically binds to membrane bound IgM of a B-cellReceptor Complex comprising: (a) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 5; (b) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 6;(c) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 7; (d) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 8; (e) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:9;and (f) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:
 10. 55. A monoclonal antibody or antigen bindingfragment thereof that specifically binds membrane bound IgM of a B-cellReceptor Complex comprising a heavy chain variable region and a lightchain variable region, wherein: (a) the heavy chain variable region (VH)is encoded by the nucleic acid sequence of SEQ ID NO: 1; and (b) thelight chain variable region (VL) is encoded by the nucleic acid sequenceof SEQ ID NO:
 3. 56. A recombinant nucleic acid comprising the nucleicacid sequences of SEQ ID NO: 1 and SEQ ID NO: 3
 57. A recombinantpolypeptide comprising the amino acid sequences of SEQ ID NO: 2 and SEQID NO:
 4. 58. A hybridoma cell line selected from the group consistingof a hybridoma cell line designated ATCC PTA-121719, a hybridoma cellline designated ATCC PTA-121717, a hybridoma cell line designated ATCCPTA-121718, and a hybridoma cell line designated ATCC PTA-121716.